Modulators of atp-binding cassette transporters

ABSTRACT

Compounds of the present invention, and pharmaceutically acceptable compositions thereof, are useful as modulators of ATP-Binding Cassette (“ABC”) transporters or fragments thereof, including Cystic Fibrosis Transmembrane Conductance Regulator (“CFTR”). The present invention also relates to methods of treating ABC transporter mediated diseases using compounds of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuing application of and claims thebenefit of priority under 35 U.S.C. §120 of co-pending InternationalApplication Serial No. PCT/US06/043289, filed Nov. 8, 2006, which claimsthe benefit, under 35 U.S.C. §119, of U.S. provisional patentapplication Ser. Nos. 60/734,506, filed on Nov. 8, 2005; 60/754,086,filed on Dec. 27, 2005; and 60/802,458, filed on May 22, 2006, theentire contents of each of above applications is incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to modulators of ATP-Binding Cassette(“ABC”) transporters or fragments thereof, including Cystic FibrosisTransmembrane Conductance Regulator (“CFTR”), compositions thereof, andmethods therewith. The present invention also relates to methods oftreating ABC transporter mediated diseases using such modulators.

BACKGROUND OF THE INVENTION

ABC transporters are a family of membrane transporter proteins thatregulate the transport of a wide variety of pharmacological agents,potentially toxic drugs, and xenobiotics, as well as anions. ABCtransporters are homologous membrane proteins that bind and use cellularadenosine triphosphate (ATP) for their specific activities. Some ofthese transporters were discovered as multi-drug resistance proteins(like the MDR1-P glycoprotein, or the multi-drug resistance protein,MRP1), defending malignant cancer cells against chemotherapeutic agents.To date, 48 ABC Transporters have been identified and grouped into 7families based on their sequence identity and function.

ABC transporters regulate a variety of important physiological roleswithin the body and provide defense against harmful environmentalcompounds. Because of this, they represent important potential drugtargets for the treatment of diseases associated with defects in thetransporter, prevention of drug transport out of the target cell, andintervention in other diseases in which modulation of ABC transporteractivity may be beneficial.

One member of the ABC transporter family commonly associated withdisease is the cAMP/ATP-mediated anion channel, CFTR. CFTR is expressedin a variety of cells types, including absorptive and secretoryepithelia cells, where it regulates anion flux across the membrane, aswell as the activity of other ion channels and proteins. In epitheliacells, normal functioning of CFTR is critical for the maintenance ofelectrolyte transport throughout the body, including respiratory anddigestive tissue. CFTR is composed of approximately 1480 amino acidsthat encode a protein made up of a tandem repeat of transmembranedomains, each containing six transmembrane helices and a nucleotidebinding domain. The two transmembrane domains are linked by a large,polar, regulatory (R)-domain with multiple phosphorylation sites thatregulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in CysticFibrosis (“CF”), the most common fatal genetic disease in humans. CysticFibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia leads to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, >1000 disease causingmutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl⁻ channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

In addition to Cystic Fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. These include, but are not limited to, chronic obstructivepulmonary disease (COPD), dry eye disease, and Sjögren's Syndrome.

COPD is characterized by airflow limitation that is progressive and notfully reversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such asCystic Fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Modulators of CFTR activity mayhydrate the various organs afflicted by the disease and help to elevatethe associated symptoms.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of ABC transporters bythe ER machinery has been shown to be the underlying basis not only forCF disease, but for a wide range of other isolated and inheriteddiseases. The two ways that the ER machinery can malfunction is eitherby loss of coupling to ER export of the proteins leading to degradation,or by the ER accumulation of these defective/misfolded proteins [AridorM, et al., Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al.,Neurochem. International, 43, pp 1-7 (2003); Rutishauser, J., et al.,Swiss Med Wkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21,pp. 466-469 (2000); Bross P., et al., Human Mut., 14, pp. 186-198(1999)]. The diseases associated with the first class of ER malfunctionare Cystic fibrosis (due to misfolded ΔF508-CFTR as discussed above),Hereditary emphysema (due to a1-antitrypsin; non Piz variants),Hereditary hemochromatosis, Coagulation-Fibrinolysis deficiencies, suchas Protein C deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses (due to Lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-Hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus (due to Insulinreceptor), Laron dwarfism (due to Growth hormone receptor),Myleoperoxidase deficiency, Primary hypoparathyroidism (due toPreproparathyroid hormone), Melanoma (due to Tyrosinase). The diseasesassociated with the latter class of ER malfunction are Glycanosis CDGtype 1, Hereditary emphysema (due to α1-Antitrypsin (PiZ variant),Congenital hyperthyroidism, Osteogenesis imperfecta (due to Type I, II,IV procollagen), Hereditary hypofibrinogenemia (due to Fibrinogen), ACTdeficiency (due to α1-Antichymotrypsin), Diabetes insipidus (DI),Neurophyseal DI (due to Vasopvessin hormone/V2-receptor), Neprogenic DI(due to Aquaporin II), Charcot-Marie Tooth syndrome (due to Peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, Amyotrophic lateral sclerosis, Progressivesupranuclear plasy, Pick's disease, several polyglutamine neurologicaldisorders a such as Huntington, Spinocerebullar ataxia type I, Spinaland bulbar muscular atrophy, Dentatorubal pallidoluysian, and Myotonicdystrophy, as well as Spongiform encephalopathies, such as HereditaryCreutzfeldt-Jakob disease (due to Prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A) andStraussler-Scheinker syndrome (due to Prp processing defect).

In addition to up-regulation of CFTR activity, reducing anion secretionby CFTR modulators may be beneficial for the treatment of secretorydiarrheas, in which epithelial water transport is dramatically increasedas a result of secretagogue activated chloride transport. The mechanisminvolves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the major consequencesof diarrheal diseases, resulting from excessive chloride transport arecommon to all, and include dehydration, acidosis, impaired growth anddeath.

Acute and chronic diarrheas represent a major medical problem in manyareas of the world. Diarrhea is both a significant factor inmalnutrition and the leading cause of death (5,000,000 deaths/year) inchildren less than five years old.

Secretory diarrheas are also a dangerous condition in patients ofacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). 16 million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Diarrhea in barn animals and pets such as cows, pigs, and horses, sheep,goats, cats and dogs, also known as scours, is a major cause of death inthese animals. Diarrhea can result from any major transition, such asweaning or physical movement, as well as in response to a variety ofbacterial or viral infections and generally occurs within the first fewhours of the animal's life.

The most common diarrhea causing bacteria is enterotoxogenic E-coli(ETEC) having the K99 pilus antigen. Common viral causes of diarrheainclude rotavirus and coronavirus. Other infectious agents includecryptosporidium, giardia lamblia, and salmonella, among others.

Symptoms of rotaviral infection include excretion of watery feces,dehydration and weakness. Coronavirus causes a more severe illness inthe newborn animals, and has a higher mortality rate than rotaviralinfection. Often, however, a young animal may be infected with more thanone virus or with a combination of viral and bacterial microorganisms atone time. This dramatically increases the severity of the disease.

Accordingly, there is a need for modulators of an ABC transporteractivity, and compositions thereof, that can be used to modulate theactivity of the ABC transporter in the cell membrane of a mammal.

There is a need for methods of treating ABC transporter mediateddiseases using such modulators of ABC transporter activity.

There is a need for methods of modulating an ABC transporter activity inan ex vivo cell membrane of a mammal.

There is a need for modulators of CFTR activity that can be used tomodulate the activity of CFTR in the cell membrane of a mammal.

There is a need for methods of treating CFTR-mediated diseases usingsuch modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are useful asmodulators of ABC transporter activity. These compounds have the generalformula (I):

or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R′₃,R₄, and n are described herein.

These compounds and pharmaceutically acceptable compositions are usefulfor treating or lessening the severity of a variety of diseases,disorders, or conditions, including, but not limited to, cysticfibrosis, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, DiabetesMellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, hereditaryemphysema, congenital hyperthyroidism, osteogenesis imperfecta,hereditary hypofibrinogenemia, ACT deficiency, Diabetes Insipidus (DI),neurophyseal DI, neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders a such as Huntington, spinocerebullar ataxia typeI, spinal and bulbar muscular atrophy, dentatorubal pallidoluysian, andmyotonic dystrophy, as well as spongiform encephalopathies, such ashereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, and Sjogren'sdisease.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cft/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing,e.g. activity, by a measurable amount. Compounds that modulate ABCTransporter activity, such as CFTR activity, by increasing the activityof the ABC Transporter, e.g., a CFTR anion channel, are called agonists.Compounds that modulate ABC Transporter activity, such as CFTR activity,by decreasing the activity of the ABC Transporter, e.g., CFTR anionchannel, are called antagonists. An agonist interacts with an ABCTransporter, such as CFTR anion channel, to increase the ability of thereceptor to transduce an intracellular signal in response to endogenousligand binding. An antagonist interacts with an ABC Transporter, such asCFTR, and competes with the endogenous ligand(s) or substrate(s) forbinding site(s) on the receptor to decrease the ability of the receptorto transduce an intracellular signal in response to endogenous ligandbinding.

The phrase “treating or reducing the severity of an ABC Transportermediated disease” refers both to treatments for diseases that aredirectly caused by ABC Transporter and/or CFTR activities andalleviation of symptoms of diseases not directly caused by ABCTransporter and/or CFTR anion channel activities. Examples of diseaseswhose symptoms may be affected by ABC Transporter and/or CFTR activityinclude, but are not limited to, Cystic fibrosis, Hereditary emphysema,Hereditary hemochromatosis, Coagulation-Fibrinolysis deficiencies, suchas Protein C deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism,Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma,Glycanosis CDG type 1, Hereditary emphysema, Congenital hyperthyroidism,Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders a such as Huntington,Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,Dentatorubal pallidoluysian, and Myotonic dystrophy, as well asSpongiform encephalopathies, such as Hereditary Creutzfeldt-Jakobdisease, Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eyedisease, and Sjogren's disease.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausolito: 1999, and “March'sAdvanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J.,John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J.,John Wiley & Sons, New York: 2001.

As used herein the term “aliphatic’ encompasses the terms alkyl,alkenyl, alkynyl, each of which being optionally substituted as setforth below.

As used herein, an “alkyl” group refers to a saturated aliphatichydrocarbon group containing 1-8 (e.g., 1-6 or 1-4) carbon atoms. Analkyl group can be straight or branched. Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-heptyl, or2-ethylhexyl. An alkyl group can be substituted (i.e., optionallysubstituted) with one or more substituents such as halo, cycloaliphatic[e.g., cycloalkyl or cycloalkenyl], heterocycloaliphatic [e.g.,heterocycloalkyl or heterocycloalkenyl], aryl, heteroaryl, alkoxy,aroyl, heteroaroyl, acyl [e.g., (aliphatic)carbonyl,(cycloaliphatic)carbonyl, or (heterocycloaliphatic)carbonyl], nitro,cyano, amido [e.g., (cycloalkylalkyl)carbonylamino, arylcarbonylamino,aralkylcarbonylamino, (heterocycloalkyl)carbonylamino,(heterocycloalkylalkyl)carbonylamino, heteroarylcarbonylamino,heteroaralkylcarbonylamino], amino [e.g., aliphaticamino,cycloaliphaticamino, or heterocycloaliphaticamino], sulfonyl [e.g.,aliphaticsulfonyl], sulfinyl, sulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, carboxy, carbamoyl, cycloaliphaticoxy,heterocycloaliphaticoxy, aryloxy, heteroaryloxy, aralkyloxy,heteroarylalkoxy, alkoxycarbonyl, alkylcarbonyloxy, or hydroxy. Withoutlimitation, some examples of substituted alkyls include carboxyalkyl(such as HOOC-alkyl, alkoxycarbonylalkyl, and alkylcarbonyloxyalkyl),cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, hydroxyalkyl, aralkyl,(alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as(alkylsulfonylamino)alkyl), aminoalkyl, amidoalkyl,(cycloaliphatic)alkyl, cyanoalkyl, or haloalkyl.

As used herein, an “alkenyl” group refers to an aliphatic carbon groupthat contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and at least onedouble bond. Like an alkyl group, an alkenyl group can be straight orbranched. Examples of an alkenyl group include, but are not limited to,allyl, isoprenyl, 2-butenyl, and 2-hexenyl. An alkenyl group can beoptionally substituted with one or more substituents such as halo,cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy, aroyl,heteroaroyl, acyl [e.g., (cycloaliphatic)carbonyl, or(heterocycloaliphatic)carbonyl], nitro, cyano, acyl [e.g.,aliphaticcarbonyl, cycloaliphaticcarbonyl, arylcarbonyl,heterocycloaliphaticcarbonyl or heteroarylcarbonyl], amido [e.g.,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino alkylaminocarbonyl,cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl,arylaminocarbonyl, or heteroarylaminocarbonyl], amino [e.g.,aliphaticamino, or aliphaticsulfonylamino], sulfonyl [e.g.,alkylsulfonyl, cycloaliphaticsulfonyl, or arylsulfonyl], sulfinyl,sulfanyl, sulfoxy, urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy,carbamoyl, cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy,heteroaryloxy, aralkyloxy, heteroarylalkoxy, alkoxycarbonyl,alkylcarbonyloxy, or hydroxy.

As used herein, an “alkynyl” group refers to an aliphatic carbon groupthat contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and has at least onetriple bond. An alkynyl group can be straight or branched. Examples ofan alkynyl group include, but are not limited to, propargyl and butynyl.An alkynyl group can be optionally substituted with one or moresubstituents such as aroyl, heteroaroyl, alkoxy, cycloalkyloxy,heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy, nitro, carboxy,cyano, halo, hydroxy, sulfo, mercapto, sulfanyl [e.g., aliphaticsulfanylor cycloaliphaticsulfanyl], sulfinyl [e.g., aliphaticsulfinyl orcycloaliphaticsulfinyl], sulfonyl [e.g., aliphaticsulfonyl,aliphaticaminosulfonyl, or cycloaliphaticsulfonyl], amido [e.g.,aminocarbonyl, alkylaminocarbonyl, alkylcarbonylamino,cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl,cycloalkylcarbonylamino, arylaminocarbonyl, arylcarbonylamino,aralkylcarbonylamino, (heterocycloalkyl)carbonylamino,(cycloalkylalkyl)carbonylamino, heteroaralkylcarbonylamino,heteroarylcarbonylamino or heteroarylaminocarbonyl], urea, thiourea,sulfamoyl, sulfamide, alkoxycarbonyl, alkylcarbonyloxy, cycloaliphatic,heterocycloaliphatic, aryl, heteroaryl, acyl [e.g.,(cycloaliphatic)carbonyl or (heterocycloaliphatic)carbonyl], amino[e.g., aliphaticamino], sulfoxy, oxo, carboxy, carbamoyl,(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, or (heteroaryl)alkoxy.

As used herein, an “amido” encompasses both “aminocarbonyl” and“carbonylamino” These terms when used alone or in connection withanother group refers to an amido group such as N(R^(X)R^(Y))—C(O)— orR^(Y)C(O)—N(R^(X))— when used terminally and —C(O)—N(R^(X))— or—N(R^(X))—C(O)— when used internally, wherein R^(X) and R^(Y) aredefined below. Examples of amido groups include alkylamido (such asalkylcarbonylamino or alkylcarbonylamino), (heterocycloaliphatic)amido,(heteroaralkyl)amido, (heteroaryl)amido, (heterocycloalkyl)alkylamido,arylamido, aralkylamido, (cycloalkyl)alkylamido, or cycloalkylamido.

As used herein, an “amino” group refers to —NR^(X)R^(Y) wherein each ofR^(X) and R^(Y) is independently hydrogen, alkyl, cycloaliphatic,(cycloaliphatic)aliphatic, aryl, araliphatic, heterocycloaliphatic,(heterocycloaliphatic)aliphatic, heteroaryl, carboxy, sulfanyl,sulfinyl, sulfonyl, (aliphatic)carbonyl, (cycloaliphatic)carbonyl,((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or(heteroaraliphatic)carbonyl, each of which being defined herein andbeing optionally substituted. Examples of amino groups includealkylamino, dialkylamino, or arylamino. When the term “amino” is not theterminal group (e.g., alkylcarbonylamino), it is represented by—NR^(X)—. R^(X) has the same meaning as defined above.

As used herein, an “aryl” group used alone or as part of a larger moietyas in “aralkyl”, “aralkoxy”, or “aryloxyalkyl” refers to monocyclic(e.g., phenyl); bicyclic (e.g., indenyl, naphthalenyl,tetrahydronaphthyl, tetrahydroindenyl); and tricyclic (e.g., fluorenyltetrahydrofluorenyl, or tetrahydroanthracenyl, anthracenyl) ring systemsin which the monocyclic ring system is aromatic or at least one of therings in a bicyclic or tricyclic ring system is aromatic. The bicyclicand tricyclic ring systems include benzofused 2-3 membered carbocyclicrings. For example, a benzofused group includes phenyl fused with two ormore C₄₋₈ carbocyclic moieties. An aryl is optionally substituted withone or more substituents including aliphatic [e.g., alkyl, alkenyl, oralkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic;heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl; heteroaryl;alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy;heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl;heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring of abenzofused bicyclic or tricyclic aryl); nitro; carboxy; amido; acyl[e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl;((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl;(heterocycloaliphatic)carbonyl;((heterocycloaliphatic)aliphatic)carbonyl; or(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl oraminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl orcycloaliphaticsulfinyl]; sulfanyl [e.g., aliphaticsulfanyl]; cyano;halo; hydroxy; mercapto; sulfoxy; urea; thiourea; sulfamoyl; sulfamide;or carbamoyl. Alternatively, an aryl can be unsubstituted.

Non-limiting examples of substituted aryls include haloaryl [e.g.,mono-, di (such as p, m-dihaloaryl), and (trihalo)aryl]; (carboxy)aryl[e.g., (alkoxycarbonyl)aryl, ((aralkyl)carbonyloxy)aryl, and(alkoxycarbonyl)aryl]; (amido)aryl [e.g., (aminocarbonyl)aryl,(((alkylamino)alkyl)aminocarbonyl)aryl, (alkylcarbonyl)aminoaryl,(arylaminocarbonyl)aryl, and (((heteroaryl)amino)carbonyl)aryl];aminoaryl [e.g., ((alkylsulfonyl)amino)aryl or ((dialkyl)amino)aryl];(cyanoalkyl)aryl; (alkoxy)aryl; (sulfamoyl)aryl [e.g.,(aminosulfonyl)aryl]; (alkylsulfonyl)aryl; (cyano)aryl;(hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl; (hydroxy)aryl,((carboxy)alkyl)aryl; (((dialkyl)amino)alkyl)aryl; (nitroalkyl)aryl;(((alkylsulfonyl)amino)alkyl)aryl; ((heterocycloaliphatic)carbonyl)aryl;((alkylsulfonyl)alkyl)aryl; (cyanoalkyl)aryl; (hydroxyalkyl)aryl;(alkylcarbonyl)aryl; alkylaryl; (trihaloalkyl)aryl;p-amino-m-alkoxycarbonylaryl; p-amino-m-cyanoaryl; p-halo-m-aminoaryl;or (m-(heterocycloaliphatic)-o-(alkyl))aryl.

As used herein, an “araliphatic” such as an “aralkyl” group refers to analiphatic group (e.g., a C₁₋₄ alkyl group) that is substituted with anaryl group. “Aliphatic,” “alkyl,” and “aryl” are defined herein. Anexample of an araliphatic such as an aralkyl group is benzyl.

As used herein, an “aralkyl” group refers to an alkyl group (e.g., aC₁₋₄ alkyl group) that is substituted with an aryl group. Both “alkyl”and “aryl” have been defined above. An example of an aralkyl group isbenzyl. An aralkyl is optionally substituted with one or moresubstituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl,including carboxyalkyl, hydroxyalkyl, or haloalkyl such astrifluoromethyl], cycloaliphatic [e.g., cycloalkyl or cycloalkenyl],(cycloalkyl)alkyl, heterocycloalkyl, (heterocycloalkyl)alkyl, aryl,heteroaryl, alkoxy, cycloalkyloxy, heterocycloalkyloxy, aryloxy,heteroaryloxy, aralkyloxy, heteroaralkyloxy, aroyl, heteroaroyl, nitro,carboxy, alkoxycarbonyl, alkylcarbonyloxy, amido [e.g., aminocarbonyl,alkylcarbonylamino, cycloalkylcarbonylamino,(cycloalkylalkyl)carbonylamino, arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, or heteroaralkylcarbonylamino], cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, a “bicyclic ring system” includes 8-12 (e.g., 9, 10, or11) membered structures that form two rings, wherein the two rings haveat least one atom in common (e.g., 2 atoms in common). Bicyclic ringsystems include bicycloaliphatics (e.g., bicycloalkyl orbicycloalkenyl), bicycloheteroaliphatics, bicyclic aryls, and bicyclicheteroaryls.

As used herein, a “cycloaliphatic” group encompasses a “cycloalkyl”group and a “cycloalkenyl” group, each of which being optionallysubstituted as set forth below.

As used herein, a “cycloalkyl” group refers to a saturated carbocyclicmono- or bicyclic (fused or bridged) ring of 3-10 (e.g., 5-10) carbonatoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, cubyl,octahydro-indenyl, decahydro-naphthyl, bicyclo[3.2.1]octyl,bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.2.]decyl,bicyclo[2.2.2]octyl, adamantyl, azacycloalkyl, or((aminocarbonyl)cycloalkyl)cycloalkyl. A “cycloalkenyl” group, as usedherein, refers to a non-aromatic carbocyclic ring of 3-10 (e.g., 4-8)carbon atoms having one or more double bonds. Examples of cycloalkenylgroups include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl,cyclooctenyl, hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl,cyclopentenyl, bicyclo[2.2.2]octenyl, or bicyclo[3.3.1]nonenyl. Acycloalkyl or cycloalkenyl group can be optionally substituted with oneor more substituents such as aliphatic [e.g., alkyl, alkenyl, oralkynyl], cycloaliphatic, (cycloaliphatic) aliphatic,heterocycloaliphatic, (heterocycloaliphatic) aliphatic, aryl,heteroaryl, alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy,aryloxy, heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,(cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino,(aryl)carbonylamino, (araliphatic)carbonylamino,(heterocycloaliphatic)carbonylamino,((heterocycloaliphatic)aliphatic)carbonylamino,(heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro,carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g.,(cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, or(heteroaraliphatic)carbonyl], cyano, halo, hydroxy, mercapto, sulfonyl[e.g., alkylsulfonyl and arylsulfonyl], sulfinyl [e.g., alkylsulfinyl],sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea, sulfamoyl,sulfamide, oxo, or carbamoyl.

As used herein, “cyclic moiety” includes cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl, each of which has beendefined previously.

As used herein, the term “heterocycloaliphatic” encompasses aheterocycloalkyl group and a heterocycloalkenyl group, each of whichbeing optionally substituted as set forth below.

As used herein, a “heterocycloalkyl” group refers to a 3-10 memberedmono- or bicylic (fused or bridged) (e.g., 5- to 10-membered mono- orbicyclic) saturated ring structure, in which one or more of the ringatoms is a heteroatom (e.g., N, O, S, or combinations thereof). Examplesof a heterocycloalkyl group include piperidyl, piperazyl,tetrahydropyranyl, tetrahydrofuryl, 1,4-dioxolanyl, 1,4-dithianyl,1,3-dioxolanyl, oxazolidyl, isoxazolidyl, morpholinyl, thiomorpholyl,octahydrobenzofuryl, octahydrochromenyl, octahydrothiochromenyl,octahydroindolyl, octahydropyrindinyl, decahydroquinolinyl,octahydrobenzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl,1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, and2,6-dioxa-tricyclo[3.3.1.0^(3,7)]nonyl. A monocyclic heterocycloalkylgroup can be fused with a phenyl moiety such as tetrahydroisoquinoline.A “heterocycloalkenyl” group, as used herein, refers to a mono- orbicylic (e.g., 5- to 10-membered mono- or bicyclic) non-aromatic ringstructure having one or more double bonds, and wherein one or more ofthe ring atoms is a heteroatom (e.g., N, O, or S). Monocyclic andbicycloheteroaliphatics are numbered according to standard chemicalnomenclature.

A heterocycloalkyl or heterocycloalkenyl group can be optionallysubstituted with one or more substituents such as aliphatic [e.g.,alkyl, alkenyl, or alkynyl], cycloaliphatic, (cycloaliphatic)aliphatic,heterocycloaliphatic, (heterocycloaliphatic)aliphatic, aryl, heteroaryl,alkoxy, (cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy,heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,(cycloaliphatic)carbonylamino, ((cycloaliphatic)aliphatic)carbonylamino, (aryl)carbonylamino,(araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino,((heterocycloaliphatic) aliphatic)carbonylamino,(heteroaryl)carbonylamino, or (heteroaraliphatic)carbonylamino], nitro,carboxy [e.g., HOOC—, alkoxycarbonyl, or alkylcarbonyloxy], acyl [e.g.,(cycloaliphatic)carbonyl, ((cycloaliphatic) aliphatic)carbonyl,(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,((heterocycloaliphatic)aliphatic)carbonyl, or(heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy, mercapto,sulfonyl [e.g., alkylsulfonyl or arylsulfonyl], sulfinyl [e.g.,alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

A “heteroaryl” group, as used herein, refers to a monocyclic, bicyclic,or tricyclic ring system having 4 to 15 ring atoms wherein one or moreof the ring atoms is a heteroatom (e.g., N, O, S, or combinationsthereof) and in which the monocyclic ring system is aromatic or at leastone of the rings in the bicyclic or tricyclic ring systems is aromatic.A heteroaryl group includes a benzofused ring system having 2 to 3rings. For example, a benzofused group includes benzo fused with one ortwo 4 to 8 membered heterocycloaliphatic moieties (e.g., indolizyl,indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl,benzo[b]thiophenyl, quinolinyl, or isoquinolinyl). Some examples ofheteroaryl are azetidinyl, pyridyl, 1H-indazolyl, furyl, pyrrolyl,thienyl, thiazolyl, oxazolyl, imidazolyl, tetrazolyl, benzofuryl,isoquinolinyl, benzthiazolyl, xanthene, thioxanthene, phenothiazine,dihydroindole, benzo[1,3]dioxole, benzo[b]furyl, benzo[b]thiophenyl,indazolyl, benzimidazolyl, benzthiazolyl, puryl, cinnolyl, quinolyl,quinazolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl, isoquinolyl,4H-quinolizyl, benzo-1,2,5-thiadiazolyl, or 1,8-naphthyridyl.

Without limitation, monocyclic heteroaryls include furyl, thiophenyl,2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl, pyrazolyl,isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl, 4-H-pyranyl,pyridyl, pyridazyl, pyrimidyl, pyrazolyl, pyrazyl, or 1,3,5-triazyl.Monocyclic heteroaryls are numbered according to standard chemicalnomenclature.

Without limitation, bicyclic heteroaryls include indolizyl, indolyl,isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl, benzo[b]thiophenyl,quinolinyl, isoquinolinyl, indolizyl, isoindolyl, indolyl,benzo[b]furyl, bexo[b]thiophenyl, indazolyl, benzimidazyl,benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl, isoquinolyl, cinnolyl,phthalazyl, quinazolyl, quinoxalyl, 1,8-naphthyridyl, or pteridyl.Bicyclic heteroaryls are numbered according to standard chemicalnomenclature.

A heteroaryl is optionally substituted with one or more substituentssuch as aliphatic [e.g., alkyl, alkenyl, or alkynyl]; cycloaliphatic;(cycloaliphatic)aliphatic; heterocycloaliphatic;(heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy;(cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy; heteroaryloxy;(araliphatic)oxy; (heteroaraliphatic)oxy; aroyl; heteroaroyl; amino; oxo(on a non-aromatic carbocyclic or heterocyclic ring of a bicyclic ortricyclic heteroaryl); carboxy; amido; acyl [e.g., aliphaticcarbonyl;(cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl;(araliphatic)carbonyl; (heterocycloaliphatic)carbonyl;((heterocycloaliphatic)aliphatic)carbonyl; or(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl oraminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g.,aliphaticsulfanyl]; nitro; cyano; halo; hydroxy; mercapto; sulfoxy;urea; thiourea; sulfamoyl; sulfamide; or carbamoyl. Alternatively, aheteroaryl can be unsubstituted.

Non-limiting examples of substituted heteroaryls include(halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl];(carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl]; cyanoheteroaryl;aminoheteroaryl [e.g., ((alkylsulfonyl)amino)heteroaryl and((dialkyl)amino)heteroaryl]; (amido)heteroaryl [e.g.,aminocarbonylheteroaryl, ((alkylcarbonyl)amino)heteroaryl,((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl,(((heteroaryl)amino)carbonyl)heteroaryl,((heterocycloaliphatic)carbonyl)heteroaryl, and((alkylcarbonyl)amino)heteroaryl]; (cyanoalkyl)heteroaryl;(alkoxy)heteroaryl; (sulfamoyl)heteroaryl [e.g.,(aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g.,(alkylsulfonyl)heteroaryl]; (hydroxyalkyl)heteroaryl;(alkoxyalkyl)heteroaryl; (hydroxy)heteroaryl;((carboxy)alkyl)heteroaryl; [((dialkyl)amino)alkyl]heteroaryl;(heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl;(nitroalkyl)heteroaryl; (((alkylsulfonyl)amino)alkyl)heteroaryl;((alkylsulfonyl)alkyl)heteroaryl; (cyanoalkyl)heteroaryl;(acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl]; (alkyl)heteroaryl,and (haloalkyl)heteroaryl [e.g., trihaloalkylheteroaryl].

A “heteroaraliphatic” (such as a heteroaralkyl group) as used herein,refers to an aliphatic group (e.g., a C₁₋₄ alkyl group) that issubstituted with a heteroaryl group. “Aliphatic,” “alkyl,” and“heteroaryl” have been defined above.

A “heteroaralkyl” group, as used herein, refers to an alkyl group (e.g.,a C₁₋₄ alkyl group) that is substituted with a heteroaryl group. Both“alkyl” and “heteroaryl” have been defined above. A heteroaralkyl isoptionally substituted with one or more substituents such as alkyl(including carboxyalkyl, hydroxyalkyl, and haloalkyl such astrifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl,heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy,cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy,heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl,alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino,cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino,arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, “cyclic moiety” includes cycloalkyl, heterocycloalkyl,cycloalkenyl, heterocycloalkenyl, aryl, or heteroaryl, each of which hasbeen defined previously.

As used herein, an “acyl” group refers to a formyl group or R^(X)—C(O)—(such as -alkyl-C(O)—, also referred to as “alkylcarbonyl”) where R^(X)and “alkyl” have been defined previously. Acetyl and pivaloyl areexamples of acyl groups.

As used herein, an “aroyl” or “heteroaroyl” refers to an aryl-C(O)— or aheteroaryl-C(O)—. The aryl and heteroaryl portion of the aroyl orheteroaroyl is optionally substituted as previously defined.

As used herein, an “alkoxy” group refers to an alkyl-O— group where“alkyl” has been defined previously.

As used herein, a “carbamoyl” group refers to a group having thestructure —O—CO—NR^(X)R^(Y) or —NR^(X)—OO—O—R^(Z) wherein R^(X) andR^(Y) have been defined above and R^(Z) can be aliphatic, aryl,araliphatic, heterocycloaliphatic, heteroaryl, or heteroaraliphatic.

As used herein, a “carboxy” group refers to —COOH, —COOR^(X), —OC(O)H,—OC(O)R^(X) when used as a terminal group; or —OC(O)— or —C(O)O— whenused as an internal group.

As used herein, a “haloaliphatic” group refers to an aliphatic groupsubstituted with 1, 2, or 3 halogen. For instance, the term haloalkylincludes the group —CF₃.

As used herein, a “mercapto” group refers to —SH.

As used herein, a “sulfo” group refers to —SO₃H or —SO₃R^(X) when usedterminally or —S(O)₃— when used internally.

As used herein, a “sulfamide” group refers to the structure—NR^(X)—S(O)₂—NR^(Y)R^(Z) when used terminally and —NR^(X)—S(O)₂—NR^(Y)—when used internally, wherein R^(X), R^(Y), and R^(Z) have been definedabove.

As used herein, a “sulfamoyl” group refers to the structure—S(O)₂—NR^(X)R^(Y) or —NR^(X)—S(O)₂—R^(Z) when used terminally; or—S(O)₂—NR^(X)— or —NR^(X)—S(O)₂— when used internally, wherein R^(X),R^(Y), and R^(Z) are defined above.

As used herein a “sulfanyl” group refers to —S—R^(X) when usedterminally and —S— when used internally, wherein R^(X) has been definedabove. Examples of sulfanyls include alkylsulfanyl.

As used herein a “sulfinyl” group refers to —S(O)—R^(X) when usedterminally and —S(O)— when used internally, wherein R^(X) has beendefined above.

As used herein, a “sulfonyl” group refers to —S(O)₂—R^(X) when usedterminally and —S(O)₂— when used internally, wherein R^(X) has beendefined above.

As used herein, a “sulfoxy” group refers to —O—SO—R^(X) or —SO—O—R^(X),when used terminally and —O—S(O)— or —S(O)—O— when used internally,where R^(X) has been defined above.

As used herein, a “halogen” or “halo” group refers to fluorine,chlorine, bromine or iodine.

As used herein, an “alkoxycarbonyl,” which is encompassed by the termcarboxy, used alone or in connection with another group refers to agroup such as alkyl-O—C(O)—-.

As used herein, an “alkoxyalkyl” refers to an alkyl group such asalkyl-O-alkyl-, wherein alkyl has been defined above.

As used herein, a “carbonyl” refer to —C(O)—.

As used herein, an “oxo” refers to ═O.

As used herein, an “aminoalkyl” refers to the structure(R^(X)R^(Y))N-alkyl-.

As used herein, a “cyanoalkyl” refers to the structure (NC)-alkyl-.

As used herein, a “urea” group refers to the structure—NR^(X)—CO—NR^(Y)R^(Z) and a “thiourea” group refers to the structure—NR^(X)—CS—NR^(Y)R^(Z) when used terminally and —NR^(x)—CO—NR^(Y)— or—NR^(X)—CS—NR^(Y)— when used internally, wherein R^(X), R^(Y), and R^(Z)have been defined above.

As used herein, a “guanidino” group refers to the structure —N═C(N(R^(X)R^(Y)))N(R^(X)R^(Y)) wherein R^(X) and R^(Y) have been defined above.

As used herein, the term “amidino” group refers to the structure—C═(NR^(X))N(R^(X)R^(Y)) wherein R^(X) and R^(Y) have been definedabove.

In general, the term “vicinal” refers to the placement of substituentson a group that includes two or more carbon atoms, wherein thesubstituents are attached to adjacent carbon atoms.

In general, the term “geminal” refers to the placement of substituentson a group that includes two or more carbon atoms, wherein thesubstituents are attached to the same carbon atom.

The terms “terminally” and “internally” refer to the location of a groupwithin a substituent. A group is terminal when the group is present atthe end of the substituent not further bonded to the rest of thechemical structure. Carboxyalkyl, i.e., R^(X)O(O)C-alkyl is an exampleof a carboxy group used terminally. A group is internal when the groupis present in the middle of a substituent to at the end of thesubstituent bound to the rest of the chemical structure. Alkylcarboxy(e.g., alkyl-C(O)O— or alkyl-OC(O)—) and alkylcarboxyaryl (e.g.,alkyl-C(O)O-aryl- or alkyl-O(CO)-aryl-) are examples of carboxy groupsused internally.

As used herein, the term “amidino” group refers to the structure—C═(NR^(X))N(R^(X)R^(Y)) wherein R^(X) and e have been defined above.

As used herein, “cyclic group” includes mono-, bi-, and tri-cyclic ringsystems including cycloaliphatic, heterocycloaliphatic, aryl, orheteroaryl, each of which has been previously defined.

As used herein, a “bridged bicyclic ring system” refers to a bicyclicheterocyclicalipahtic ring system or bicyclic cycloaliphatic ring systemin which the rings are bridged. Examples of bridged bicyclic ringsystems include, but are not limited to, adamantanyl, norbornanyl,bicyclo[3.2.1]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl,bicyclo[3.2.3]nonyl, 2-oxa-bicyclo[2.2.2]octyl,1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, and2,6-dioxa-tricyclo[3.3.1.03,7]nonyl. A bridged bicyclic ring system canbe optionally substituted with one or more substituents such as alkyl(including carboxyalkyl, hydroxyalkyl, and haloalkyl such astrifluoromethyl), alkenyl, alkynyl, cycloalkyl, (cycloalkyl)alkyl,heterocycloalkyl, (heterocycloalkyl)alkyl, aryl, heteroaryl, alkoxy,cycloalkyloxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, aralkyloxy,heteroaralkyloxy, aroyl, heteroaroyl, nitro, carboxy, alkoxycarbonyl,alkylcarbonyloxy, aminocarbonyl, alkylcarbonylamino,cycloalkylcarbonylamino, (cycloalkylalkyl)carbonylamino,arylcarbonylamino, aralkylcarbonylamino,(heterocycloalkyl)carbonylamino, (heterocycloalkylalkyl)carbonylamino,heteroarylcarbonylamino, heteroaralkylcarbonylamino, cyano, halo,hydroxy, acyl, mercapto, alkylsulfanyl, sulfoxy, urea, thiourea,sulfamoyl, sulfamide, oxo, or carbamoyl.

As used herein, an “aliphatic chain” refers to a branched or straightaliphatic group (e.g., alkyl groups, alkenyl groups, or alkynyl groups).A straight aliphatic chain has the structure —[CH₂]_(v)—, where v is1-6. A branched aliphatic chain is a straight aliphatic chain that issubstituted with one or more aliphatic groups. A branched aliphaticchain has the structure —[CHQ]_(v)— where Q is hydrogen or an aliphaticgroup; however, Q shall be an aliphatic group in at least one instance.The term aliphatic chain includes alkyl chains, alkenyl chains, andalkynyl chains, where alkyl, alkenyl, and alkynyl are defined above.

The phrase “optionally substituted” is used interchangeably with thephrase “substituted or unsubstituted.” As described herein, compounds ofthe invention can optionally be substituted with one or moresubstituents, such as are illustrated generally above, or as exemplifiedby particular classes, subclasses, and species of the invention. Asdescribed herein, the variables R₁, R₂, R₃, and R₄, and other variablescontained therein formulae I encompass specific groups, such as alkyland aryl. Unless otherwise noted, each of the specific groups for thevariables R₁, R₂, R₃, and R₄, and other variables contained therein canbe optionally substituted with one or more substituents describedherein. Each substituent of a specific group is further optionallysubstituted with one to three of halo, cyano, oxoalkoxy, hydroxy, amino,nitro, aryl, haloalkyl, and alkyl. For instance, an alkyl group can besubstituted with alkylsulfanyl and the alkylsulfanyl can be optionallysubstituted with one to three of halo, cyano, oxoalkoxy, hydroxy, amino,nitro, aryl, haloalkyl, and alkyl. As an additional example, thecycloalkyl portion of a (cycloalkyl)carbonylamino can be optionallysubstituted with one to three of halo, cyano, alkoxy, hydroxy, nitro,haloalkyl, and alkyl. When two alkoxy groups are bound to the same atomor adjacent atoms, the two alkoxy groups can form a ring together withthe atom(s) to which they are bound.

In general, the term “substituted,” whether preceded by the term“optionally” or not, refers to the replacement of hydrogen radicals in agiven structure with the radical of a specified substituent. Specificsubstituents are described above in the definitions and below in thedescription of compounds and examples thereof. Unless otherwiseindicated, an optionally substituted group can have a substituent ateach substitutable position of the group, and when more than oneposition in any given structure can be substituted with more than onesubstituent selected from a specified group, the substituent can beeither the same or different at every position. A ring substituent, suchas a heterocycloalkyl, can be bound to another ring, such as acycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings shareone common atom. As one of ordinary skill in the art will recognize,combinations of substituents envisioned by this invention are thosecombinations that result in the formation of stable or chemicallyfeasible compounds.

The phrase “up to”, as used herein, refers to zero or any integer numberthat is equal or less than the number following the phrase. For example,“up to 3” means any one of 0, 1, 2, and 3.

The phrase “stable or chemically feasible,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and preferablytheir recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

As used herein, an effective amount is defined as the amount required toconfer a therapeutic effect on the treated patient, and is typicallydetermined based on age, surface area, weight, and condition of thepatient. The interrelationship of dosages for animals and humans (basedon milligrams per meter squared of body surface) is described byFreireich et al., Cancer Chemother. Rep., 50: 219 (1966). Body surfacearea may be approximately determined from height and weight of thepatient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley,New York, 537 (1970). As used herein, “patient” refers to a mammal,including a human.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention. Additionally, unless otherwise stated, structuresdepicted herein are also meant to include compounds that differ only inthe presence of one or more isotopically enriched atoms. For example,compounds having the present structures except for the replacement ofhydrogen by deuterium or tritium, or the replacement of a carbon by a¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Suchcompounds are useful, for example, as analytical tools or probes inbiological assays.

Compounds

Compounds of the present invention are useful modulators of ABCtransporters and are useful in the treatment of ABC transport mediateddiseases.

A. Generic Compounds

The present invention includes a compound of formula (I),

or a pharmaceutically acceptable salt thereof, wherein:

Each R₁ is an optionally substituted C₁₋₆ aliphatic, an optionallysubstituted aryl, an optionally substituted heteroaryl, an optionallysubstituted C₃₋₁₀ cycloaliphatic, an optionally substituted 3 to 10membered heterocycloaliphatic, carboxy [e.g., hydroxycarbonyl oralkoxycarbonyl], amido [e.g., aminocarbonyl], amino, halo, or hydroxy;

provided that at least one R₁ is an optionally substitutedcycloaliphatic, an optionally substituted heterocycloaliphatic, anoptionally substituted aryl, or an optionally substituted heteroarylattached to the 5- or 6-position of the pyridyl ring;

Each R₂ is hydrogen, an optionally substituted C₁₋₆ aliphatic, anoptionally substituted C₃₋₆ cycloaliphatic, an optionally substitutedphenyl, or an optionally substituted heteroaryl;

Each R₃ and R′₃ together with the carbon atom to which they are attachedform an optionally substituted C₃₋₇ cycloaliphatic or an optionallysubstituted heterocycloaliphatic;

Each R₄ is an optionally substituted aryl or an optionally substitutedheteroaryl; and

Each n is 1, 2, 3 or 4.

In another aspect, the present invention includes compounds of formula(I′):

or a pharmaceutically acceptable salt thereof,

wherein:

one of G₁ and G₂ is a nitrogen, and the other is a carbon; and

R₁, R₂, R₃, R′₃, R₄, and n are defined above.

Specific Embodiments A. Substituent R₁

Each R₁ is independently an optionally substituted C₁₋₆ aliphatic, anoptionally substituted aryl, an optionally substituted heteroaryl, anoptionally substituted C₃₋₁₀ membered cycloaliphatic, an optionallysubstituted 3 to 10 membered heterocycloaliphatic, carboxy [e.g.,hydroxycarbonyl or alkoxycarbonyl], amido [e.g., aminocarbonyl], amino,halo, or hydroxy.

In some embodiments, one R₁ is an optionally substituted C₁₋₆ aliphatic.In several examples, one R₁ is an optionally substituted C₁₋₆ alkyl, anoptionally substituted C₂₋₆ alkenyl, or an optionally substituted C₂₋₆alkynyl. In several examples, one R₁ is C₁₋₆ alkyl, C₂₋₆ alkenyl, orC₂₋₆ alkynyl.

In several embodiments, one R₁ is an aryl or heteroaryl with 1, 2, or 3substituents. In several examples, one R₁ is a monocyclic aryl orheteroaryl. In several embodiments, R₁ is an aryl or heteroaryl with 1,2, or 3 substituents. In several examples, R₁ is a monocyclic aryl orheteroaryl.

In several embodiments, at least one R₁ is an optionally substitutedaryl or an optionally substituted heteroaryl and R₁ is bonded to thecore structure at the 6 position on the pyridine ring.

In several embodiments, at least one R₁ is an optionally substitutedaryl or an optionally substituted heteroaryl and R₁ is bonded to thecore structure at the 5 position on the pyridine ring.

In several embodiments, one R₁ is phenyl with up to 3 substituents. Inseveral embodiments, R₁ is phenyl with up to 3 substituents.

In several embodiments, one R₁ is a heteroaryl ring with up to 3substituents. In certain embodiments, one R₁ is a monocyclic heteroarylring with up to 3 substituents. In other embodiments, one R₁ is abicyclic heteroaryl ring with up to 3 substituents. In severalembodiments, R₁ is a heteroaryl ring with up to 3 substituents. Incertain embodiments, R₁ is a monocyclic heteroaryl ring with up to 3substituents. In other embodiments, R₁ is a bicyclic heteroaryl ringwith up to 3 substituents.

In several embodiments, one R₁ is carboxy [e.g., hydroxycarbonyl oralkoxycarbonyl]. Or, one R₁ is amido [e.g., aminocarbonyl]. Or, one R₁is amino. Or, is halo. Or, is cyano. Or, hydroxyl.

In some embodiments, R₁ is hydrogen, methyl, ethyl, i-propyl, t-butyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, allyl, F, Cl, methoxy,ethoxy, i-propoxy, t-butoxy, CF₃, OCF₃, CN, hydroxyl, or amino. Inseveral examples, R₁ is hydrogen, methyl, methoxy, F, CF₃ or OCF₃. Inseveral examples, R₁ can be hydrogen. Or, R₁ can be methyl. Or, R₁ canbe CF₃. Or, R₁ can be methoxy.

In several embodiments, R₁ is substituted with no more than threesubstituents selected from halo, oxo, or optionally substitutedaliphatic, cycloaliphatic, heterocycloaliphatic, amino [e.g.,(aliphatic)amino], amido [e.g., aminocarbonyl,((aliphatic)amino)carbonyl, and ((aliphatic)₂-amino)carbonyl], carboxy[e.g., alkoxycarbonyl and hydroxycarbonyl], sulfamoyl [e.g.,aminosulfonyl, ((aliphatic)₂amino)sulfonyl,((cycloaliphatic)aliphatic)aminosulfonyl, and((cycloaliphatic)amino)sulfonyl], cyano, alkoxy, aryl, heteroaryl [e.g.,monocyclic heteroaryl and bicycloheteroaryl], sulfonyl [e.g.,aliphaticsulfonyl or (heterocycloaliphatic)sulfonyl], sulfinyl [e.g.,aliphaticsulfinyl], aroyl, heteroaroyl, or heterocycloaliphaticcarbonyl.

In several embodiments, R₁ is substituted with halo. Examples of R₁substituents include F, Cl, and Br. In several examples, R₁ issubstituted with F.

In several embodiments, R₁ is substituted with an optionally substitutedaliphatic. Examples of R₁ substituents include optionally substitutedalkoxyaliphatic, heterocycloaliphatic, aminoalkyl, hydroxyalkyl,(heterocycloalkyl)aliphatic, alkylsulfonylaliphatic,alkylsulfonylaminoaliphatic, alkylcarbonylaminoaliphatic,alkylaminoaliphatic, or alkylcarbonylaliphatic.

In several embodiments, R₁ is substituted with an optionally substitutedamino Examples of R₁ substituents include aliphaticcarbonylamino,aliphaticamino, arylamino, or aliphaticsulfonylamino.

In several embodiments, R₁ is substituted with a sulfonyl. Examples ofR₁ substituents include heterocycloaliphaticsulfonyl, aliphaticsulfonyl, aliphaticaminosulfonyl, aminosulfonyl,aliphaticcarbonylaminosulfonyl, alkoxyalkylheterocycloalkylsulfonyl,alkylheterocycloalkylsulfonyl, alkylaminosulfonyl,cycloalkylaminosulfonyl, (heterocycloalkyl)alkylaminosulfonyl, andheterocycloalkylsulfonyl.

In several embodiments, R₁ is substituted with carboxy. Examples of R₁substituents include alkoxycarbonyl and hydroxycarbonyl.

In several embodiments R₁ is substituted with amido. Examples of R₁substituents include alkylaminocarbonyl, aminocarbonyl,((aliphatic)₂amino)carbonyl, and[((aliphatic)aminoaliphatic)amino]carbonyl.

In several embodiments, R₁ is substituted with carbonyl. Examples of R₁substituents include arylcarbonyl, cycloaliphaticcarbonyl,heterocycloaliphaticcarbonyl, and heteroarylcarbonyl.

In some embodiments, R₁ is hydrogen. In some embodiments, R₁ is—Z^(A)R₅, wherein each Z^(A) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(A) are optionally and independently replaced by —CO—,—CS—, —CONR^(A)—, —CONR^(A)NR^(A)—, —CO₂—, —OCO—, —NR^(A)CO₂—, —O—,—NR^(A)CONR^(A)—, —OCONR^(A)—, —NR^(A)NR^(A)—, —NR^(A)CO—, —S—, —SO—,—SO₂—, —NR^(A)—, —SO₂NR^(A)—, —NR^(A)SO₂—, or —NR^(A)SO₂NR^(A)—. Each R₅is independently R^(A), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃. EachR^(A) is independently a C₁₋₈ aliphatic group, a cycloaliphatic, aheterocycloaliphatic, an aryl, or a heteroaryl, each of which isoptionally substituted with 1, 2, or 3 of R^(D). Each R^(D) is —Z^(D)R₉,wherein each Z^(D) is independently a bond or an optionally substitutedbranched or straight C₁₋₆ aliphatic chain wherein up to two carbon unitsof Z^(D) are optionally and independently replaced by —CO—, —CS—,—CONR^(E)—, —CONR^(E)NR^(E)—, —CO₂—, —OCO—, —NR^(E)CO₂—, —O—,—NR^(E)CONR^(E)—, —OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—,—SO₂—, —NR^(E)—, —SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—. Each R₉is independently R^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃. EachR^(E) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In some embodiments, each R^(D) is independently —Z^(D)R₉; wherein eachZ^(D) can independently be a bond or an optionally substituted branchedor straight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D)are optionally and independently replaced by —O—, —NHC(O)—,—C(O)NR^(E)—, —SO₂—, —NHSO₂—, —NHC(O)—, —NR^(E)SO₂—, —SO₂NH—,—SO₂NR^(E)—, —NH—, or —C(O)O—. In some embodiments, one carbon unit ofZ^(D) is replaced by —O—. Or, by —NHC(O)—. Or, by —C(O)NR^(E)—. Or, by—SO₂—. Or, by —NHSO₂—. Or, by —NHC(O)—. Or, by —SO—. Or, by —NR^(E)SO₂—.Or, by —SO₂NH—. Or, by —SO₂NR^(E)—. Or, by —NH—. Or, by —C(O)O—.

In some embodiments, R₉ is hydrogen. In some embodiments, R₉ isindependently an optionally substituted aliphatic. In some embodiments,R₉ is an optionally substituted cycloaliphatic. Or, is an optionallysubstituted heterocycloaliphatic. Or, is an optionally substituted aryl.Or, is an optionally substituted heteroaryl. Or, halo.

In some embodiments, one R₁ is aryl or heteroaryl, each optionallysubstituted with 1, 2, or 3 of R^(D), wherein R^(D) is defined above.

In several embodiments, one R₁ is carboxy [e.g., hydroxycarbonyl oralkoxycarbonyl]. Or, one R₁ is amido [e.g., aminocarbonyl]. Or, one R₁is amino Or, is halo. Or, is cyano. Or, hydroxyl.

In some embodiments, one R₁ that is attached to 5- or 6-position of thepyridyl ring is aryl or heteroaryl, each optionally substituted with 1,2, or 3 of R^(D), wherein R^(D) is defined above. In some embodiments,the one R₁ attached to the 5- or 6-position of the pyridyl ring isphenyl optionally substituted with 1, 2, or 3 of R^(D), wherein R^(D) isdefined above. In some embodiments, the one R₁ attached to the 5- or6-position of the pyridyl ring is heteroaryl optionally substituted with1, 2, or 3 of R^(D). In several embodiments, the one R₁ attached to the5- or 6-position of the pyridyl ring is 5 or 6 membered heteroarylhaving 1, 2, or 3 heteroatom independently selected from the groupconsisting of oxygen, nitrogen and sulfur. In other embodiments, the 5or 6 membered heteroaryl is substituted with 1 R^(D).

In some embodiments, one R₁ attached to the 5- or 6-position of thepyridyl ring is a phenyl substituted with 1 R^(D). In some embodiments,one R₁ attached to the 5- or 6-position of the pyridyl ring is a phenylsubstituted with 2 R^(D). In some embodiments, one R₁ attached to the 5-or 6-position of the pyridyl ring is a phenyl substituted with 3 R^(D).

In several embodiments, R₁ is:

wherein

W₁ is —C(O)—, —SO₂—, or —CH₂—;

D is H, hydroxyl, or an optionally substituted group selected fromaliphatic, cycloaliphatic, alkoxy, and amino; and

R^(D) is defined above.

In several embodiments, W₁ is —C(O)—. Or, W₁ is —SO₂—. Or, W₁ is —CH₂—.

In several embodiments, D is OH. Or, D is an optionally substituted C₁₋₆aliphatic or an optionally substituted C₃-C₈ cycloaliphatic. Or, D is anoptionally substituted alkoxy. Or, D is an optionally substituted amino

In several examples, D is

wherein each of A and B is independently H, an optionally substitutedC₁₋₆ aliphatic, an optionally substituted C₃-C₈ cycloaliphatic, or

A and B, taken together, form an optionally substituted 3-7 memberedheterocycloaliphatic ring.

In several embodiments, A is H and B is an optionally substituted C₁₋₆aliphatic. In several embodiments, B is substituted with 1, 2, or 3substituents. Or, both, A and B, are H. Exemplary substituents includeoxo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, dialkyamino, oran optionally substituted group selected from cycloaliphatic,heterocycloaliphatic, aryl, and heteroaryl.

In several embodiments, A is H and B is an optionally substituted C₁₋₆aliphatic. Or, both, A and B, are H. Exemplary substituents include oxo,alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, and an optionallysubstituted heterocycloaliphatic.

In several embodiments, B is C₁₋₆ alkyl, optionally substituted withoxo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, or an optionallysubstituted group selected from cycloaliphatic, heterocycloaliphatic,aryl, and heteroaryl. In several embodiments, B is substituted with oxo,C₁₋₆ alkyl, hydroxy, hydroxy-(C₁₋₆)alkyl, (C₁₋₆)alkoxy,(C₁₋₆)alkoxy(C₁₋₆)alkyl, C₃₋₈ cycloaliphatic, 3-8 memberedheterocycloaliphatic, phenyl, and 5-10 membered heteroaryl. In oneexample, B is C₁₋₆ alkyl substituted with optionally substituted phenyl.

In several embodiments, A and B, taken together, form an optionallysubstituted 3-7 membered heterocycloaliphatic ring. In several examples,the heterocycloaliphatic ring is optionally substituted with 1, 2, or 3substituents. Exemplary such rings include optionally substitutedpyrrolidinyl, piperidinyl, morpholinyl, and piperazinyl. Exemplarysubstituents on such rings include halo, oxo, alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, acyl (e.g., alkylcarbonyl), amino,amido, and carboxy. In some embodiments, the substituent is halo, oxo,alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, amino, amido, orcarboxy.

In several embodiments, R^(D) is hydrogen, halo, or an optionallysubstituted group selected from aliphatic, cycloaliphatic, amino,hydroxy, alkoxy, carboxy, amido, carbonyl, cyano, aryl, or heteroaryl.In several examples, R^(D) is hydrogen, halo, an optionally substitutedC₁₋₆ aliphatic, or an optionally substituted alkoxy. In severalexamples, R^(D) is hydrogen, F, Cl, an optionally substituted C₁₋₆alkyl, or an optionally substituted —O(C₁₋₆ alkyl). Examples of R^(D)include hydrogen, F, Cl, methyl, ethyl, i-propyl, t-butyl, —OMe, —OEt,1-propoxy, t-butoxy, CF₃, or —OCF₃. In some examples, R^(D) is hydrogen,F, methyl, methoxy, CF₃, or —OCF₃. R^(D) can be hydrogen. R^(D) can beF. R^(D) can be methyl. R^(D) can be methoxy.

In several embodiments, R₁ is:

wherein:

W₁ is —C(O)—, —SO₂—, or —CH₂—;

Each of A and B is independently H, an optionally substituted C₁₋₆aliphatic, an optionally substituted C₃-C₈ cycloaliphatic; or

A and B, taken together, form an optionally substituted 3-7 memberedheterocycloaliphatic ring.

In some embodiments, one R₁ that is attached to the 5- or 6-position ofthe pyridyl ring is cycloaliphatic or heterocycloaliphatic, eachoptionally substituted with 1, 2, or 3 of R^(D); wherein R^(D) is—Z^(D)R₉; wherein each Z^(D) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(D) are optionally and independently replaced by —CO—,—CS—, —CONR^(E)—, —CONR^(E)NR^(E)—, —CO₂—, —OCO—, NR^(E)CO₂—, —O—,—NR^(E)CONR^(E)—, —OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—,—SO₂—, —NR^(E)—, —SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—; each R₉is independently R^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃; andeach R^(E) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In several examples, one R₁ that is attached to the 5- or 6-position ofthe pyridyl ring is an optionally substituted C₃-C₈ cycloaliphatic.

In some embodiments, one R₁ that is attached to the 5- or 6-position ofthe pyridyl ring is an optionally substituted C₃-C₈ cycloalkyl or anoptionally substituted C₃-C₈ cycloalkenyl.

In several embodiments, one R₁ that is attached to the 5- or 6-positionof the pyridyl ring is C₃-C₈ cycloalkyl or C₃-C₈ cycloalkenyl. Examplesof cycloalkyl and cycloalkenyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, andcycloheptenyl.

In some embodiments, R1 is:

In several examples, R₁ is one selected from:

B. Substituent R₂

Each R₂ can be hydrogen. Each R₂ can be an optionally substituted groupselected from C₁₋₆ aliphatic, C₃₋₆ cycloaliphatic, phenyl, andheteroaryl.

In several embodiments, R₂ is a C₁₋₆ aliphatic optionally substitutedwith 1, 2, or 3 halo, C₁₋₂ aliphatic, or alkoxy. In several examples, R₂can be substituted methyl, ethyl, propyl, or butyl. In several examples,R₂ can be methyl, ethyl, propyl, or butyl.

In several embodiments, R₂ is hydrogen.

C. Substituents R₃ and R′₃

Each R₃ and R′₃ together with the carbon atom to which they are attachedform a C₃₋₇ cycloaliphatic or a heterocycloaliphatic, each of which isoptionally substituted with 1, 2, or 3 substituents.

In several embodiments, R₃ and R′₃ together with the carbon atom towhich they are attached form a C₃₋₇ cycloaliphatic or a C₃₋₇heterocycloaliphatic, each of which is optionally substituted with 1, 2,or 3 of —Z^(B)R₇, wherein each Z^(B) is independently a bond, or anoptionally substituted branched or straight C₁₋₄ aliphatic chain whereinup to two carbon units of Z^(B) are optionally and independentlyreplaced by —CO—, —CS—, —CONR^(B)—, —CONR^(B)NR^(B)—, —CO₂—, —OCO—,—NR^(B)CO₂—, —O—, —NR^(B)CONR^(B)—, —OCONR^(B)—, —NR^(B)NR^(B)—,—NR^(B)CO—, —S—, —SO—, —SO₂NR^(B)—, —NR^(B)SO₂—, or —NR^(B)SO₂NR^(B)—;each R₇ is independently R^(B), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or—OCF₃; and each R^(B) is independently hydrogen, an optionallysubstituted C₁₋₈ aliphatic group, an optionally substitutedcycloaliphatic, an optionally substituted heterocycloaliphatic, anoptionally substituted aryl, or an optionally substituted heteroaryl.

In several embodiments, R₃ and R′₃ together with the carbon atom towhich they are attached form a 3, 4, 5, or 6 membered cycloaliphaticthat is optionally substituted with 1, 2, or 3 substituents. In severalexamples, R₃, R′₃, and the carbon atom to which they are attached forman optionally substituted cyclopropyl group. In several alternativeexamples, R₃, R′₃, and the carbon atom to which they are attached forman optionally substituted cyclobutyl group. In several other examples,R₃, R′₃, and the carbon atom to which they are attached form anoptionally substituted cyclopentyl group. In other examples, R₃, R′₃,and the carbon atom to which they are attached form an optionallysubstituted cyclohexyl group. In more examples, R₃ and R′₃ together withthe carbon atom to which they are attached form an unsubstitutedcyclopropyl.

In several embodiments, R₃ and R′₃ together with the carbon atom towhich they are attached form a 5, 6, or 7 membered optionallysubstituted heterocycloaliphatic. In other examples, R₃, R′₃, and thecarbon atom to which they are attached form an optionally substitutedtetrahydropyranyl group.

In some embodiments, R₃ and R′₃ together with the carbon atom to whichthey are attached form an unsubstituted C₃₋₇ cycloaliphatic or anunsubstituted heterocycloaliphatic. In several examples, R₃ and R′₃together with the carbon atom to which they are attached form anunsubstituted cyclopropyl, an unsubstituted cyclopentyl, or anunsubstituted cyclohexyl.

D. Substituent R₄

Each R₄ is independently an optionally substituted aryl or an optionallysubstituted heteroaryl.

In several embodiments, R₄ is an aryl having 6 to 10 members (e.g., 7 to10 members) optionally substituted with 1, 2, or 3 substituents.Examples of R₄ include optionally substituted benzene, naphthalene, orindene. Or, examples of R₄ can be optionally substituted phenyl,optionally substituted naphthyl, or optionally substituted indenyl.

In several embodiments, R₄ is an optionally substituted heteroaryl.Examples of R₄ include monocyclic and bicyclic heteroaryl, such abenzofused ring system in which the phenyl is fused with one or two 4-8membered heterocycloaliphatic groups.

In some embodiments, R₄ is an aryl or heteroaryl, each optionallysubstituted with 1, 2, or 3 of —Z^(C)R₈. In some embodiments, R₄ is anaryl optionally substituted with 1, 2, or 3 of —Z^(C)R₈. In someembodiments, R₄ is phenyl optionally substituted with 1, 2, or 3 of—Z^(C)R₈. Or, R₄ is a heteroaryl optionally substituted with 1, 2, or 3of —Z^(C)R₈. Each Z^(C) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(C) are optionally and independently replaced by —CO—,—CS—, —CONR^(C)—, —CONR^(C)NR^(C)—, —CO₂—, —OCO—, —NR^(C)CO₂—, —O—,—NR^(C)CONR^(C)—, —OCONR^(C)—, —NR^(C)NR^(C)—, —NR^(C)CO—, —S—, —SO—,—SO₂—, —NR^(S)—, —SO₂NR^(C)—, —NR^(C)SO₂—, or —NR^(C)SO₂NR^(C)—. Each R₈is independently R^(C), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃. EachR^(C) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In some embodiments, two occurrences of —Z^(C)R₈, taken together withcarbons to which they are attached, form a 4-8 membered saturated,partially saturated, or aromatic ring with up to 3 ring atomsindependently selected from the group consisting of O, NH, NR^(C), andS; wherein R^(C) is defined herein.

In several embodiments, R₄ is one selected from

E. Exemplary Compound Families

In several embodiments, R₁ is an optionally substituted cyclic groupthat is attached to the core structure at the 5 or 6 position of thepyridine ring.

In several examples, R₁ is an optionally substituted aryl that isattached to the 5 position of the pyridine ring. In other examples, R₁is an optionally substituted aryl that is attached to the 6 position ofthe pyridine ring.

In more examples, R₁ is an optionally substituted heteroaryl that isattached to the 5 position of the pyridine ring. In still otherexamples, R₁ is an optionally substituted heteroaryl that is attached tothe 6 position of the pyridine ring.

In other embodiments, R₁ is an optionally substituted cycloaliphatic oran optionally substituted heterocycloaliphatic that is attached to thepyridine ring at the 5 or 6 position.

Accordingly, another aspect of the present invention provides compoundsof formula (II):

or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R′₃,and R₄ are defined in formula I.

In some embodiments, each R₁ is aryl or heteroaryl optionallysubstituted with 1, 2, or 3 of R^(D), wherein R^(D) is —Z^(D)R₉, whereineach Z^(D) is independently a bond or an optionally substituted branchedor straight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D)are optionally and independently replaced by —CO—, —CS—, —CONR^(E)—,—CONR^(E)NR^(E)—, —CO₂—, —OCO—, —NR^(E)CO₂—, —O—, —NR^(E)CONR^(E)—,—OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—, —SO₂—, —NR^(E)—,—SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—; each R₉ is independentlyR^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃; each R^(E) isindependently hydrogen, an optionally substituted C₁₋₈ aliphatic group,an optionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl.

In some embodiment, each R₁ is cycloaliphatic or heterocycloaliphaticoptionally substituted with 1, 2, or 3 of R^(D); wherein R^(D) isdefined above.

Another aspect of the present invention provides compounds of formula(III):

or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R′₃,and R₄ are defined in formula I.

In some embodiments, each R₁ is aryl or heteroaryl optionallysubstituted with 1, 2, or 3 of R^(D), wherein R^(D) is —Z^(D)R₉, whereineach Z^(D) is independently a bond or an optionally substituted branchedor straight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(D)are optionally and independently replaced by —CO—, —CS—, —CONR^(E)—,—CONR^(E)NR^(E)—, —CO₂—, —OCO—, —NR^(E)CO₂—, —O—, —NR^(E)CONR^(E)—,—OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—, —SO₂—, —NR^(E)—,—SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—; each R₉ is independentlyR^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃; each R^(E) isindependently hydrogen, an optionally substituted C₁₋₈ aliphatic group,an optionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl.

In some embodiments, each R₁ is cycloaliphatic or heterocycloaliphaticoptionally substituted with 1, 2, or 3 of R^(D); wherein R^(D) isdefined above.

In another aspect, the present invention includes compounds of formula(IV):

or a pharmaceutically acceptable salt thereof, wherein R₂, R₃, R′₃, andR₄ are defined in formula I.

R^(D) is —Z^(D)R₉; wherein each Z^(D) is independently a bond or anoptionally substituted branched or straight C₁₋₆ aliphatic chain whereinup to two carbon units of Z^(D) are optionally and independentlyreplaced by —CO—, —CS—, —CONR^(E)—, —CONR^(E)NR^(E)—, —CO₂—, —OCO—,—NR^(E)CO₂—, —O—, —NR^(E)CONR^(E)—, —OCONR^(E)—, —NR^(E)NR^(E)—,—NR^(E)CO—, —S—, —SO—, —SO₂—, —NR^(E)—, —SO₂NR^(E)—, —NR^(E)SO₂—, or—NR^(E)SO₂NR^(E)—.

R₉ is independently R^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃.

Each R^(E) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In several embodiments, Z^(D) is independently a bond or is anoptionally substituted branched or straight C₁₋₆ aliphatic chain whereinone carbon unit of Z^(D) is optionally replaced by —SO₂—, —CONR^(E)—,—NR^(E)SO₂—, or —SO₂NR^(E)—. For example, Z^(D) is an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein one carbonunit of Z^(D) is optionally replaced by —SO₂—. In other examples, R₉ isan optionally substituted heteroaryl or an optionally substitutedheterocycloaliphatic. In additional examples, R₉ is an optionallysubstituted heterocycloaliphatic having 1-2 nitrogen atoms, and R₉attaches directly to —SO₂— via a ring nitrogen.

In another aspect, the present invention includes compounds of formulaV-A or formula V-B:

or a pharmaceutically acceptable salt thereof,

wherein:

T is an optionally substituted C₁₋₂ aliphatic chain, wherein each of thecarbon units is optionally and independently replaced by —CO—, —CS—,—COCO—, —SO₂—, —B(OH)—, or —B(O(C₁₋₆ alkyl))-;

Each of R₁′ and R₁″ is independently a bond or an optionally substitutedC₁₋₆ aliphatic, an optionally substituted aryl, an optionallysubstituted heteroaryl, an optionally substituted 3 to 10 memberedcycloaliphatic, an optionally substituted 3 to 10 memberedheterocycloaliphatic, carboxy, amido, amino, halo, or hydroxy;

R^(D1) is attached to carbon 3″ or 4″;

each R^(D1) and R^(D2) is —Z^(D)R₉, wherein each Z^(D) is independentlya bond or an optionally substituted branched or straight C₁₋₆ aliphaticchain wherein up to two carbon units of Z^(D) are optionally andindependently replaced by —CO—, —CS—, —CONR^(E)—, —CONR^(E)NR^(E)—,—CO₂—, —OCO—, —NR^(E)CO₂—, —O—, —NR^(E)CONR^(E)—, —OCONR^(E)—,—NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—, —SO₂—, —NR^(E)—, —SO₂NR^(E)—,—NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—;

R₉ is independently R^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃;

or R^(D1) and R^(D2), taken together with atoms to which they areattached, form a 3-8 membered saturated, partially unsaturated, oraromatic ring with up to 3 ring members independently selected from thegroup consisting of O, NH, NR^(E), and S; and

each R^(E) is independently hydrogen, an optionally substituted C₁₋₈aliphatic group, an optionally substituted cycloaliphatic, an optionallysubstituted heterocycloaliphatic, an optionally substituted aryl, or anoptionally substituted heteroaryl.

In some embodiments, T is an optionally substituted —CH₂—. In some otherembodiments, T is an optionally substituted —CH₂CH₂—.

In some embodiments, T is optionally substituted by —Z^(E)R₁₀; whereineach Z^(E) is independently a bond or an optionally substituted branchedor straight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(E)are optionally and independently replaced by —CO—, —CS—, —CONR^(F)—,—CONR^(F)NR^(F)—, —CO₂—, —OCO—, —NR^(F)CO₂—, —O—, —NR^(F)CONR^(F)—,—OCONR^(F)—, —NR^(F)NR^(F)—, —NR^(F)CO—, —S—, —SO—, —SO₂—, —NR^(F)—,—SO₂NR^(F)—, —NR^(F)SO₂—, or —NR^(F)SO₂NR^(F)—; R₁₀ is independentlyR^(E), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃; each R^(E) isindependently hydrogen, an optionally substituted C₁₋₈ aliphatic group,an optionally substituted cycloaliphatic, an optionally substitutedheterocycloaliphatic, an optionally substituted aryl, or an optionallysubstituted heteroaryl. In one example, Z^(E) is —O—.

In some embodiments, R₁₀ can be an optionally substituted C₁₋₆ alkyl, anoptionally substituted C₂₋₆ alkenyl, an optionally substituted C₃₋₇cycloaliphatic, or an optionally substituted C₆₋₁₀ aryl. In oneembodiment, R₁₀ is methyl, ethyl, i-propyl, or t-butyl.

In some embodiments, up to two carbon units of T are optionallysubstituted by —CO—, —CS—, —B(OH)—, or —B(O(C₁₋₆ alkyl)-.

In some embodiments, T is selected from the group consisting of —CH₂—,—CH₂CH₂—, —CF₂—, —C(CH₃)₂—, —C(O)—,

—C(Phenyl)₂—, —B(OH)—, and —CH(OEt)-. In some embodiments, T is —CH₂—,—CF₂—, —C(CH₃)₂—,

or —C(Phenyl)₂—. In other embodiments, T is —CH₂H₂—, —C(O)—, —B(OH)—,and —CH(OEt)-. In several embodiments, T is —CH₂—, —CF₂—, —C(CH₃)₂—,

More preferably, T is —CH₂—, —CF₂—, or —C(CH₃)₂—. In severalembodiments, T is —CH₂—. Or, T is —CF₂—. Or, T is —C(CH₃)₂—.

In some embodiments, each of R₁′ and R₁″ is hydrogen. In someembodiments, each of R₁′ and R₁″ is independently —Z^(A)R₅, wherein eachZ^(A) is independently a bond or an optionally substituted branched orstraight C₁₋₆ aliphatic chain wherein up to two carbon units of Z^(A)are optionally and independently replaced by —CO—, —CS—, —CONR^(A)—,—CONR^(A)NR^(A)—, —CO₂—, —OCO—, —NR^(A)CO₂—, —O—, —NR^(A)CONR^(A)—,—OCONR^(A)—, —NR^(A)NR^(A)—, —NR^(A)CO—, —S—, —SO—, —SO₂—, —NR^(A)—,—SO₂NR^(A)—, —NR^(A)SO₂—, or —NR^(A)SO₂NR^(A)—. Each R₅ is independentlyR^(A), halo, —OH, —NH₂, —NO₂, —CN, —CF₃, or —OCF₃. Each R^(A) isindependently an optionally substituted group selected from C₁₋₈aliphatic group, a cycloaliphatic, a heterocycloaliphatic, an aryl, anda heteroaryl.

In some embodiments, R₁′ is selected from the group consisting of H,C₁₋₆ aliphatic, halo, CF₃, CHF₂, —O(C₁₋₆ aliphatic), C3-C5 cycloalkyl,or C4-C6 heterocycloalkyl containing one oxygen atom. In someembodiments, R₁′ is selected from the group consisting of H, methyl,ethyl, i-propyl, t-butyl, F. C₁, CF₃, CHF₂, —OCH₃, —OCH₂CH₃,—O-(i-propyl), or —O-(t-butyl). More preferably, R₁′ is H. Or, R₁′ ismethyl. Or, ethyl. Or, CF₃.

In some embodiments, R₁″ is selected from the group consisting of H,C₁₋₆ aliphatic, halo, CF₃, CHF₂, and —O(C₁₋₆ aliphatic). In someembodiments, R₁″ is selected from the group consisting of H, methyl,ethyl, i-propyl, t-butyl, F. C₁, CF₃, CHF₂, —OCH₃, —OCH₂CH₃,—O-(i-propyl), or —O-(t-butyl). More preferably, R₁″ is H. Or, R₁″ ismethyl. Or, ethyl. Or, CF₃.

In some embodiments, R^(D1) is attached to carbon 3″ or 4″, and is—Z^(D)R₉, wherein each Z^(D) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein up to twocarbon units of Z^(D) are optionally and independently replaced by —CO—,—CS—, —CONR^(E)—, —CONR^(E)NR^(E)—, —CO₂—, —OCO—, —NR^(E)CO₂—, —O—,—NR^(E)CONR^(E)—, —OCONR^(E)—, —NR^(E)NR^(E)—, —NR^(E)CO—, —S—, —SO—,—SO₂—, —NR^(E)—, —SO₂NR^(E)—, —NR^(E)SO₂—, or —NR^(E)SO₂NR^(E)—. In yetsome embodiments, Z^(D) is independently a bond or an optionallysubstituted branched or straight C₁₋₆ aliphatic chain wherein one carbonunit of Z^(D) is optionally replaced by —CO—, —SO—, —SO₂—, —COO—, —OCO—,—CONR^(E)—, —NR^(E)CO—, NR^(E)CO₂—, —O—, —NR^(E)SO₂—, or —SO₂NR^(E)—. Insome embodiments, one carbon unit of Z^(D) is optionally replaced by—CO—. Or, by —SO—. Or, by —SO₂—. Or, by —COO—. Or, by —OCO—. Or, by—CONR^(E)—. Or, by —NR^(E)CO—. Or, by —NR^(E)CO₂—. Or, by —O—. Or, by—NR^(E)SO₂—. Or, by —SO₂NR^(E)—.

In several embodiments, R₉ is hydrogen, halo, —OH, —NH₂, —CN, —CF₃,—OCF₃, or an optionally substituted group selected from the groupconsisting of C₁₋₆ aliphatic, C₃₋₈ cycloaliphatic, 3-8 memberedheterocycloaliphatic, C₆₋₁₀ aryl, and 5-10 membered heteroaryl. Inseveral examples, R₉ is hydrogen, F, Cl, —OH, —CN, —CF₃, or —OCF₃. Insome embodiments, R⁹ is C₁₋₆ aliphatic, C₃₋₈ cycloaliphatic, 3-8membered heterocycloaliphatic, C₆₋₁₀ aryl, and 5-10 membered heteroaryl,each of which is optionally substituted by 1 or 2 substituentsindependently selected from the group consisting of R^(E), oxo, halo,—OH, —NR^(E)R^(E), —OR^(E), —COOR^(E), and —CONR^(E)R^(E). In severalexamples, R₉ is optionally substituted by 1 or 2 substituentsindependently selected from the group consisting of oxo, F, Cl, methyl,ethyl, i-propyl, t-butyl, —CH₂OH, —CH₂CH₂OH, —C(O)OH, —C(O)NH₂,—CH₂O(C₁₋₆ alkyl), —CH₂CH₂O(C₁₋₆ alkyl), and —C(O)(C₁₋₆ alkyl).

In one embodiment, R₉ is hydrogen. In some embodiments, R₉ is selectedfrom the group consisting of C₁₋₆ straight or branched alkyl or C₂₋₆straight or branched alkenyl; wherein said alkyl or alkenyl isoptionally substituted by 1 or 2 substituents independently selectedfrom the group consisting of R^(E), oxo, halo, —OH, —NR^(E)R^(E),—OR^(E), —COOR^(E), and —CONR^(E)R^(E).

In other embodiments, R₉ is C₃₋₈ cycloaliphatic optionally substitutedby 1 or 2 substituents independently selected from the group consistingof R^(E), oxo, halo, —OH, —NR^(E)R^(E), —OR^(E), —COOR^(E), and—CONR^(E)R^(E). Examples of cycloaliphatic include but are not limitedto cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

In yet other embodiments, R₉ is a 3-8 membered heterocyclic with 1 or 2heteroatoms independently selected from the group consisting of O, NH,NR^(E), and S; wherein said heterocyclic is optionally substituted by 1or 2 substituents independently selected from the group R^(E), oxo,halo, —OH, —NR^(E)R^(E), —OR^(E), COOR^(E), and —CONR^(E)R^(E). Exampleof 3-8 membered heterocyclic include but are not limited to

In yet some other embodiments, R₉ is an optionally substituted 5-8membered heteroaryl with one or two ring atom independently selectedfrom the group consisting of O, S, and NR^(E). Examples of 5-8 memberedheteroaryl include but are not limited to

In some embodiments, R^(D1) and R^(D2), taken together with carbons towhich they are attached, form an optionally substituted 4-8 memberedsaturated, partially unsaturated, or aromatic ring with 0-2 ring atomsindependently selected from the group consisting of O, NH, NR^(E), andS. Examples of R^(D1) and R^(D2), taken together with phenyl containingcarbon atoms 3″ and 4″, include but are not limited to

In some embodiments, R^(D2) is selected from the group consisting of H,R^(E), halo, —OH, —(CH₂)_(r)NR^(E)R^(E), —(CH₂)_(r)OR^(E), —SO₂—R^(E),—NR^(E)—SO₂—R^(E), —SO₂NR^(E)R^(E), —C(O)R^(E), —C(O)OR^(E),—OC(O)OR^(E), —NR^(E)C(O)OR^(E), and —C(O)NR^(E)R^(E); wherein r is 0,1, or 2. In other embodiments, R^(D2) is selected from the groupconsisting of H, C₁₋₆ aliphatic, halo, —CN, —NH₂, —NH(C₁₋₆ aliphatic),—N(C₁₋₆ aliphatic)₂, —CH₂—N(C₁₋₆ aliphatic)₂, —CH₂—NH(C₁₋₆ aliphatic),—CH₂NH₂, —OH, —O(C₁₋₆ aliphatic), —CH₂OH, —CH₂—O(C₁₋₆ aliphatic),—SO₂(C₁₋₆ aliphatic), —N(C₁₋₆ aliphatic)-SO₂(C₁₋₆ aliphatic),—NH—SO₂(C₁₋₆ aliphatic), —SO₂NH₂, —SO₂NH(C₁₋₆ aliphatic), —SO₂N(C₁₋₆aliphatic)₂, —C(O)(C₁₋₆ aliphatic), —C(O)O(C₁₋₆ aliphatic), —C(O)OH,—OC(O)O(C₁₋₆ aliphatic), —NHC(O)(C₁₋₆ aliphatic), —NHC(O)O(C₁₋₆aliphatic), —N(C₁₋₆ aliphatic)C(O)O(C₁₋₆ aliphatic), —C(O)NH₂, and—C(O)N(C₁₋₆ aliphatic)₂. In several examples, R^(D2) is selected fromthe group consisting of H, C₁₋₆ aliphatic, halo, —CN, —NH₂, —CH₂NH₂,—OH, —O(C₁₋₆ aliphatic), —CH₂OH, —SO₂(C₁₋₆ aliphatic), —NH—SO₂(C₁₋₆aliphatic), —C(O)O(C₁₋₆ aliphatic), —C(O)OH, —NHC(O)(C₁₋₆ aliphatic),—C(O)NH₂, —C(O)NH(C₁₋₆ aliphatic), and —C(O)N(C₁₋₆ aliphatic)₂. Forexamples, R^(D2) is selected from the group consisting of H, methyl,ethyl, n-propyl, i-propyl, t-butyl, F, Cl, CN, —NH₂, —CH₂NH₂, —OH,—OCH₃, —O-ethyl, —O-(i-propyl), —O-(n-propyl), —CH₂OH, —SO₂CH₃,—NH—SO₂CH₃, —C(O)OCH₃, —C(O)OCH₂CH₃, —C(O)OH, —NHC(O)CH₃, —C(O)NH₂, and—C(O)N(CH₃)₂. In one embodiment, R^(D2) is hydrogen. In anotherembodiment, R^(D2) is methyl. Or, R^(D2) is ethyl. Or, R^(D2) is F. Or,R^(D2) is Cl. Or, —OCH₃.

In one embodiment, the present invention provides compounds of formulaVI-A-i or formula VI-A-ii:

wherein T, R^(D1), R^(D2), and R₁′ are as defined above.

In one embodiment, T is —CH₂—, —CF₂—, or —C(CH₃)₂—.

In one embodiment, R₁′ is selected from the group consisting of H, C₁₋₆aliphatic, halo, CF₃, CHF₂, —O(C₁₋₆ aliphatic), C3-C5 cycloalkyl, orC4-C6 heterocycloalkyl containing one oxygen atom. Exemplary embodimentsinclude H, methyl, ethyl, i-propyl, t-butyl, F. C₁, CF₃, CHF₂, —OCH₃,—OCH₂CH₃, —O-(i-propyl), —O-(t-butyl), cyclopropyl, or oxetanyl. Morepreferably, R₁′ is H. Or, R₁′ is methyl. Or, ethyl. Or, CF₃. Or,oxetanyl.

In one embodiment, R^(D1) is Z^(D)R₉, wherein Z^(D) is selected fromCONH, NHCO, SO₂NH, SO₂N(C₁₋₆ alkyl), NHSO₂, CH₂NHSO₂, CH₂N(CH₃)SO₂,CH₂NHCO, COO, SO₂, or CO. In one embodiment, R^(D1) is Z^(D)R₉, whereinZ^(D) is selected from CONH, SO₂NH, SO₂N(C₁₋₆ alkyl), CH₂NHSO₂,CH₂N(CH₃)SO₂, CH₂NHCO, COO, SO₂, or CO.

In one embodiment, Z^(D) is COO and R₉ is H. In one embodiment, Z^(D) isCOO and R₉ is an optionally substituted straight or branched C₁₋₆aliphatic. In one embodiment, Z^(D) is COO and R₉ is an optionallysubstituted straight or branched C₁₋₆ alkyl. In one embodiment, Z^(D) isCOO and R₉ is C₁₋₆ alkyl. In one embodiment, Z^(D) is COO and R₉ ismethyl.

In one embodiment, Z^(D) is CONH and R₉ is H. In one embodiment, Z^(D)is CONH and R₉ is an optionally substituted straight or branched C₁₋₆aliphatic. In one embodiment, Z^(D) is CONH and R₉ is straight orbranched C₁₋₆ alkyl. In one embodiment, Z^(D) is CONH and R₉ is methyl.In one embodiment, Z^(D) is CONH and R₉ is an optionally substitutedstraight or branched C₁₋₆ alkyl. In one embodiment, In one embodiment,Z^(D) is CONH and R₉ is 2-(dimethylamino)-ethyl.

In some embodiments, Z^(D) is CH₂NHCO and R₉ is an optionallysubstituted straight or branched C₁₋₆ aliphatic or an optionallysubstituted alkoxy. In some embodiments, Z^(D) is CH₂NHCO and R₉ isstraight or branched C₁₋₆ alkyl optionally substituted with halo, oxo,hydroxyl, or an optionally substituted group selected from aliphatic,cyclic, aryl, heteroaryl, alkoxy, amino, carboxyl, or carbonyl. In oneembodiment, Z^(D) is CH₂NHCO and R₉ is methyl. In one embodiment, Z^(D)is CH₂NHCO and R₉ is CF₃. In one embodiment, Z^(D) is CH₂NHCO and R₉ ist-butoxy.

In one embodiment, Z^(D) is SO₂NH and R₉ is H. In some embodiments,Z^(D) is SO₂NH and R₉ is an optionally substituted straight or branchedC₁₋₆ aliphatic. In some embodiments, Z^(D) is SO₂NH and R₉ is straightor branched C₁₋₆ alkyl optionally substituted with halo, oxo, hydroxyl,or an optionally substituted group selected from C₁₋₆ aliphatic, 3-8membered cyclic, C₆₋₁₀ aryl, 5-8 membered heteroaryl, alkoxy, amino,amido, carboxyl, or carbonyl. In one embodiment, Z^(D) is SO₂NH and R₉is methyl. In one embodiment, Z^(D) is SO₂NH and R₉ is ethyl. In oneembodiment, Z^(D) is SO₂NH and R₉ is i-propyl. In one embodiment, Z^(D)is SO₂NH and R₉ is t-butyl. In one embodiment, Z^(D) is SO₂NH and R₉ is3,3-dimethylbutyl. In one embodiment, Z^(D) is SO₂NH and R₉ is CH₂CH₂OH.In one embodiment, Z^(D) is SO₂NH and R₉ is CH(CH₃)CH₂OH. In oneembodiment, Z^(D) is SO₂NH and R₉ is CH₂CH(CH₃)OH. In one embodiment,Z^(D) is SO₂NH and R₉ is CH(CH₂OH)₂. In one embodiment, Z^(D) is SO₂NHand R₉ is CH₂CH(OH)CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH(OH)CH₂CH₃. In one embodiment, Z^(D) is SO₂NH and R₉ isC(CH₃)₂CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH(CH₂CH₃)CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH₂OCH₂CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isC(CH₃)(CH₂OH)₂. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH(OH)CH₂C(O)OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH₂N(CH₃)₂. In one embodiment, Z^(D) is SO₂NH and R₉ isCH₂CH₂NHC(O)CH₃. In one embodiment, Z^(D) is SO₂NH and R₉ isCH(CH(CH₃)₂)CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ isCH(CH₂CH₂CH₃)CH₂OH. In one embodiment, Z^(D) is SO₂NH and R₉ is1-tetrahydrofuryl-methyl. In one embodiment, Z^(D) is SO₂NH and R₉ isfurylmethyl. In one embodiment, Z^(D) is SO₂NH and R₉ is(5-methylfuryl)-methyl. In one embodiment, Z^(D) is SO₂NH and R₉ is2-pyrrolidinylethyl. In one embodiment, Z^(D) is SO₂NH and R₉ is2-(1-methylpyrrolidinyl)-ethyl. In one embodiment, Z^(D) is SO₂NH and R₉is 2-(4-morpholinyl)-ethyl. In one embodiment, Z^(D) is SO₂NH and R₉ is3-(4-morpholinyl)-propyl. In one embodiment, Z^(D) is SO₂NH and R₉ isC(CH₂CH₃)(CH₂OH)₂. In one embodiment, Z^(D) is SO₂NH and R₉ is2-(1H-imidazol-4-yl)ethyl. In one embodiment, Z^(D) is SO₂NH and R₉ is3-(1H-imidazol-1-yl)-propyl. In one embodiment, Z^(D) is SO₂NH and R₉ is2-(2-pyridinyl)-ethyl.

In some embodiment, Z^(D) is SO₂NH and R₉ is an optionally substitutedC₁₋₆ cycloaliphatic. In several examples, Z^(D) is SO₂NH and R₉ is anoptionally substituted C₁₋₆ cycloalkyl. In several examples, Z^(D) isSO₂NH and R₉ is C₁₋₆ cycloalkyl. In one embodiment, Z^(D) is SO₂NH andR₉ is cyclobutyl. In one embodiment, Z^(D) is SO₂NH and R₉ iscyclopentyl. In one embodiment, Z^(D) is SO₂NH and R₉ is cyclohexyl.

In some embodiments, Z^(D) is SO₂N(C₁₋₆ alkyl) and R₉ is an optionallysubstituted straight or branched C₁₋₆ aliphatic or an optionallysubstituted cycloaliphatic. In some embodiments, Z^(D) is SO₂N(C₁₋₆alkyl) and R₉ is an optionally substituted straight or branched C₁₋₆aliphatic. In some embodiments, Z^(D) is SO₂N(C₁₋₆ alkyl) and R₉ is anoptionally substituted straight or branched C₁₋₆ alkyl or an optionallysubstituted straight or branched C₁₋₆ alkenyl. In one embodiments, Z^(D)is SO₂N(CH₃) and R₉ is methyl. In one embodiments, Z^(D) is SO₂N(CH₃)and R₉ is n-propyl. In one embodiments, Z^(D) is SO₂N(CH₃) and R₉ isn-butyl. In one embodiments, Z^(D) is SO₂N(CH₃) and R₉ is cyclohexyl. Inone embodiments, Z^(D) is SO₂N(CH₃) and R₉ is allyl. In one embodiments,Z^(D) is SO₂N(CH₃) and R₉ is CH₂CH₂OH. In one embodiments, Z^(D) isSO₂N(CH₃) and R₉ is CH₂CH(OH)CH₂OH. In one embodiments, Z^(D) isSO₂N(CH₂CH₂CH₃) and R₉ is cyclopropylmethyl.

In one embodiment, Z^(D) is CH₂NHSO₂ and R₉ is methyl. In oneembodiment, Z^(D) is CH₂N(CH₃)SO₂ and R₉ is methyl.

In some embodiments, Z^(D) is SO₂ and R₉ is an optionally substitutedC₁₋₆ straight or branched aliphatic or an optionally substituted 3-8membered heterocyclic, having 1, 2, or 3 ring members selected from thegroup consisting of nitrogen, oxygen, sulfur, SO, or SO₂. In someembodiments, Z^(D) is SO₂ and R₉ is straight or branched C₁₋₆ alkyl or3-8 membered heterocycloaliphatic each of which is optionallysubstituted with 1, 2, or 3 of oxo, halo, hydroxyl, or an optionallysubstituted group selected from C₁₋₆ aliphatic, carbonyl, amino, andcarboxy. In one embodiment, Z^(D) is SO₂ and R₉ is methyl. In someembodiments, Z^(D) is SO₂ and examples of R₉ include

In some embodiments, R^(D2) is H, hydroxyl, halo, C₁₋₆ alkyl, C₁₋₆alkoxy, C₃₋₆ cycloalkyl, or NH₂. In several examples, R^(D2) is H, halo,C₁₋₄ alkyl, or C₁₋₄ alkoxy. Examples of R^(D2) include H, F, Cl, methyl,ethyl, and methoxy.

In some embodiments, the present invention provides compounds of formula(I′-A) or formula (I′-B):

or a pharmaceutically acceptable salt thereof,

wherein R₁, R₂, R₃, R′₃, R₄, and n are defined above.

In some embodiments, R₁ is an optionally substituted aryl. In severalexamples, R₁ is phenyl optionally substituted with 1, 2, or 3 of halo,OH, —O(C₁₋₆ aliphatic), amino, C₁₋₆ aliphatic, C₃₋₇ cycloaliphatic, 3-8membered heterocycloaliphatic, C₆₋₁₀ aryl, or 5-8 membered heteroaryl.In some embodiments, R₁ is phenyl optionally substituted with alkoxy,halo, or amino. In one embodiment, R₁ is phenyl. In one embodiment, R₁is phenyl substituted with Cl, methoxy, ethoxy, or dimethylamino.

In some embodiments, R₂ is hydrogen. In some embodiments, R₂ isoptionally substituted C₁₋₆ aliphatic.

In some embodiments, R₃, R′₃, and the carbon atom to which they areattached form an optionally substituted C₃₋₈ cycloaliphatic or anoptionally substituted 3-8 membered heterocycloaliphatic. In someembodiments, R₃, R′₃, and the carbon atom to which they are attachedform an optionally substituted C₃₋₈ cycloalkyl. In one example, R₃, R′₃,and the carbon atom to which they are attached is cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, each of which isoptionally substituted. In one example, R₃, R′₃, and the carbon atom towhich they are attached is cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl or cycloheptyl. In several examples, R₃, R′₃, and the carbonatom to which they are attached is cyclopropyl.

In some embodiments, R₄ is an optionally substituted aryl or anoptionally substituted heteroaryl. In some embodiments, R₄ is anoptionally substituted phenyl. In several embodiments, R₄ is phenylfused to a 3, 4, 5, or 6 membered heterocyclic having 1, 2, or 3 ringmembered selected from oxygen, sulfur and nitrogen. In severalembodiments, R₄ is

wherein T is defined above. In several examples, T is —CH₂—.

Alternative embodiments of R₁, R₂, R₃, R′₃, R₄, and n in formula (I′-A)or formula (I′-B) are as defined for formula (I), formula (I′), andembodiments thereof.

Exemplary compounds of the present invention include, but are notlimited to, those illustrated in Table 1 below.

TABLE 1 Examples of compounds of the present invention

1

2

3

4

5

6

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Synthetic Schemes

Compounds of the invention may be prepared by known methods or asillustrated in the examples. In one instance wherein R₁ is aryl orheteroaryl, the compounds of the invention may be prepared asillustrated in Scheme I.

Referring to Scheme I, a nitrile of formula i is alkylated (step a) witha dihalo-aliphatic in the presence of a base such as, for example, 50%sodium hydroxide and, optionally, a phase transfer reagent such as, forexample, benzyltriethylammonium chloride (BTEAC), to produce thecorresponding alkylated nitrile (not shown) which on hydrolysis producesthe acid ii. Compounds of formula II are converted to the acid chlorideiii with a suitable reagent such as, for example, thionyl chloride/DMF.Reaction of the acid chloride iii with an aminopyridine, wherein X is ahalo, of formula iv (step c) produces the amide of formula v. Reactionof the amide v with an optionally substituted boronic acid derivative(step d) in the presence of a catalyst such as, for example, palladiumacetate or dichloro-[1,1-bis(diphenylphosphino)ferrocene]palladium(II)(Pd(dppf)Cl₂), provides compounds of the invention wherein R₁ is aryl,heteroaryl, or cycloalkenyl. The boronic acid derivatives vi arecommercially available or may be prepared by known methods such asreaction of an aryl bromide with a diborane ester in the presence of acoupling reagent such as, for example, palladium acetate as described inthe examples.

In another instance where one R₁ is aryl and another R₁ is an aliphatic,alkoxy, cycloaliphatic, or heterocycloaliphatic, compounds of theinvention can be prepared as described in steps a, b, and c of Scheme Iusing an appropriately substituted aminopyridine such as

where X is halo and Q is C₁₋₆ aliphatic, aryl, heteroaryl, or 3 to 10membered cycloaliphatic or heterocycloaliphatic as a substitute for theaminopyridine of formula iv.

Formulations, Administrations, and Uses

Pharmaceutically Acceptable Compositions

Accordingly, in another aspect of the present invention,pharmaceutically acceptable compositions are provided, wherein thesecompositions comprise any of the compounds as described herein, andoptionally comprise a pharmaceutically acceptable carrier, adjuvant orvehicle. In certain embodiments, these compositions optionally furthercomprise one or more additional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative or a prodrug thereof. Accordingto the present invention, a pharmaceutically acceptable derivative or aprodrug includes, but is not limited to, pharmaceutically acceptablesalts, esters, salts of such esters, or any other adduct or derivativewhich upon administration to a patient in need is capable of providing,directly or indirectly, a compound as otherwise described herein, or ametabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington: TheScience and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy,Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, the contents of each of which isincorporated by reference herein, disclose various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by ABC transporteractivity. In certain embodiments, the present invention provides amethod of treating a condition, disease, or disorder implicated by adeficiency of ABC transporter activity, the method comprisingadministering a composition comprising a compound of formulae (I, II,III, IV, V-A, V-B, I′, I′-A, and I′-B) to a subject, preferably amammal, in need thereof.

In certain preferred embodiments, the present invention provides amethod of treating Cystic fibrosis, Hereditary emphysema, Hereditaryhemochromatosis, Coagulation-Fibrinolysis deficiencies, such as ProteinC deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism,Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma,Glycanosis CDG type 1, Hereditary emphysema, Congenital hyperthyroidism,Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders such as Huntington,Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,Dentatorubal pallidoluysian, and Myotonic dystrophy, as well asSpongiform encephalopathies, such as Hereditary Creutzfeldt-Jakobdisease (due to Prion protein processing defect), Fabry disease,Straussler-Scheinker disease, secretory diarrhea, polycystic kidneydisease, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome, comprising the step of administering to saidmammal an effective amount of a composition comprising a compound offormulae (I, II, III, IV, V-A, V-B, I′, I′-A, and I′-B), or a preferredembodiment thereof as set forth above.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising a compound of formulae (I, II, III, IV, V-A, V-B, I′, I′-A,and I′-B), or a preferred embodiment thereof as set forth above.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of Cystic fibrosis,Hereditary emphysema, Hereditary hemochromatosis,Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency,Type 1 hereditary angioedema, Lipid processing deficiencies, such asFamilial hypercholesterolemia, Type 1 chylomicronemia,Abetalipoproteinemia, Lysosomal storage diseases, such as I-celldisease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetesmellitus, Laron dwarfism, Myleoperoxidase deficiency, Primaryhypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditaryemphysema, Congenital hyperthyroidism, Osteogenesis imperfecta,Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI),Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome,Perlizaeus-Merzbacher disease, neurodegenerative diseases such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders asuch as Huntington, Spinocerebullar ataxia typeI, Spinal and bulbar muscular atrophy, Dentatorubal pallidoluysian, andMyotonic dystrophy, as well as Spongiform encephalopathies, such asHereditary Creutzfeldt-Jakob disease, Fabry disease,Straussler-Scheinker disease, secretory diarrhea, polycystic kidneydisease, chronic obstructive pulmonary disease (COPD), dry eye disease,and Sjögren's Syndrome.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of Cystic fibrosis, Hereditary emphysema, Hereditaryhemochromatosis, Coagulation-Fibrinolysis deficiencies, such as ProteinC deficiency, Type 1 hereditary angioedema, Lipid processingdeficiencies, such as Familial hypercholesterolemia, Type 1chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, suchas I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism,Myleoperoxidase deficiency, Primary hypoparathyroidism, Melanoma,Glycanosis CDG type 1, Hereditary emphysema, Congenital hyperthyroidism,Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency,Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-MarieTooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Progressive supranuclear plasy, Pick's disease,several polyglutamine neurological disorders asuch as Huntington,Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy,Dentatorubal pallidoluysian, and Myotonic dystrophy, as well asSpongiform encephalopathies, such as Hereditary Creutzfeldt-Jakobdisease, Fabry disease, Straussler-Scheinker disease, secretorydiarrhea, polycystic kidney disease, chronic obstructive pulmonarydisease (COPD), dry eye disease, and Sjögren's Syndrome.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

As described generally above, the compounds of the invention are usefulas modulators of ABC transporters. Thus, without wishing to be bound byany particular theory, the compounds and compositions are particularlyuseful for treating or lessening the severity of a disease, condition,or disorder where hyperactivity or inactivity of ABC transporters isimplicated in the disease, condition, or disorder. When hyperactivity orinactivity of an ABC transporter is implicated in a particular disease,condition, or disorder, the disease, condition, or disorder may also bereferred to as an “ABC transporter-mediated disease, condition ordisorder”. Accordingly, in another aspect, the present inventionprovides a method for treating or lessening the severity of a disease,condition, or disorder where hyperactivity or inactivity of an ABCtransporter is implicated in the disease state.

The activity of a compound utilized in this invention as a modulator ofan ABC transporter may be assayed according to methods describedgenerally in the art and in the Examples herein.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another agent used to treat the same disorder), orthey may achieve different effects (e.g., control of any adverseeffects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating ABC transporteractivity in a biological sample or a patient (e.g., in vitro or invivo), which method comprises administering to the patient, orcontacting said biological sample with a compound of formula I or acomposition comprising said compound. The term “biological sample”, asused herein, includes, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof.

Modulation of ABC transporter activity in a biological sample is usefulfor a variety of purposes that are known to one of skill in the art.Examples of such purposes include, but are not limited to, the study ofABC transporters in biological and pathological phenomena; and thecomparative evaluation of new modulators of ABC transporters.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with a compound of formulae (I, II, III, IV,V-A, V-B, I′, I′-A, and I′-B). In preferred embodiments, the anionchannel is a chloride channel or a bicarbonate channel. In otherpreferred embodiments, the anion channel is a chloride channel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional ABC transporters in amembrane of a cell, comprising the step of contacting said cell with acompound of formula (I, II, III, IV, V-A, V-B, I′, I′-A, and I′-B). Theterm “functional ABC transporter” as used herein means an ABCtransporter that is capable of transport activity. In preferredembodiments, said functional ABC transporter is CFTR.

According to another preferred embodiment, the activity of the ABCtransporter is measured by measuring the transmembrane voltagepotential. Means for measuring the voltage potential across a membranein the biological sample may employ any of the known methods in the art,such as optical membrane potential assay or other electrophysiologicalmethods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In another aspect the present invention provides a kit for use inmeasuring the activity of a ABC transporter or a fragment thereof in abiological sample in vitro or in vivo comprising (i) a compositioncomprising a compound of formula (I, II, III, IV, V-A, V-B, I′, I′-A,and I′-B) or any of the above embodiments; and (ii) instructions for a.)contacting the composition with the biological sample and b.) measuringactivity of said ABC transporter or a fragment thereof. In oneembodiment, the kit further comprises instructions for a.) contacting anadditional composition with the biological sample; b.) measuring theactivity of said ABC transporter or a fragment thereof in the presenceof said additional compound, and c.) comparing the activity of the ABCtransporter in the presence of the additional compound with the densityof the ABC transporter in the presence of a composition of formula (I,II, III, IV, V-A, V-B, I′, I′-A, and I′-B). In preferred embodiments,the kit is used to measure the density of CFTR.

Preparations and Examples General Procedure I: Carboxylic Acid BuildingBlock

Benzyltriethylammonium chloride (0.025 equivalents) and the appropriatedihalo compound (2.5 equivalents) were added to a substituted phenylacetonitrile. The mixture was heated at 70° C. and then 50% sodiumhydroxide (10 equivalents) was slowly added to the mixture. The reactionwas stirred at 70° C. for 12-24 hours to ensure complete formation ofthe cycloalkyl moiety and then heated at 130° C. for 24-48 hours toensure complete conversion from the nitrile to the carboxylic acid. Thedark brown/black reaction mixture was diluted with water and extractedwith ethyl acetate and then dichloromethane three times each to removeside products. The basic aqueous solution was acidified withconcentrated hydrochloric acid to pH less than one and the precipitatewhich began to form at pH 4 was filtered and washed with 1 Mhydrochloric acid two times. The solid material was dissolved indichloromethane and extracted two times with 1 M hydrochloric acid andone time with a saturated aqueous solution of sodium chloride. Theorganic solution was dried over sodium sulfate and evaporated to drynessto give the cycloalkylcarboxylic acid.

A. 1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid

A mixture of benzo[1,3]dioxole-5-acetonitrile (5.10 g, 31.7 mmol),1-bromo-2-chloro-ethane (9.00 mL, 109 mmol), and benzyltriethylammoniumchloride (0.181 g, 0.795 mmol) was heated at 70° C. and then 50%(wt./wt.) aqueous sodium hydroxide (26 mL) was slowly added to themixture. The reaction was stirred at 70° C. for 18 hours and then heatedat 130° C. for 24 hours. The dark brown reaction mixture was dilutedwith water (400 mL) and extracted once with an equal volume of ethylacetate and once with an equal volume of dichloromethane. The basicaqueous solution was acidified with concentrated hydrochloric acid to pHless than one and the precipitate filtered and washed with 1 Mhydrochloric acid. The solid material was dissolved in dichloromethane(400 mL) and extracted twice with equal volumes of 1 M hydrochloric acidand once with a saturated aqueous solution of sodium chloride. Theorganic solution was dried over sodium sulfate and evaporated to drynessto give a white to slightly off-white solid (5.23 g, 80%) ESI-MS m/zcalc. 206.1. found 207.1 (M+1)⁺. Retention time of 2.37 minutes. ¹H NMR(400 MHz, DMSO-d₆) δ 1.07-1.11 (m, 2H), 1.38-1.42 (m, 2H), 5.98 (s, 2H),6.79 (m, 2H), 6.88 (m, 1H), 12.26 (s, 1H).

General Procedure II: Carboxylic Acid Building Block

Sodium hydroxide (50% aqueous solution, 7.4 equivalents) was slowlyadded to a mixture of the appropriate phenyl acetonitrile,benzyltriethylammonium chloride (1.1 equivalents), and the appropriatedihalo compound (2.3 equivalents) at 70° C. The mixture was stirredovernight at 70° C. and the reaction mixture was diluted with water (30mL) and extracted with ethyl acetate. The combined organic layers weredried over sodium sulfate and evaporated to dryness to give the crudecyclopropanecarbonitrile, which was used directly in the next step.

The crude cyclopropanecarbonitrile was heated at reflux in 10% aqueoussodium hydroxide (7.4 equivalents) for 2.5 hours. The cooled reactionmixture was washed with ether (100 mL) and the aqueous phase wasacidified to pH 2 with 2M hydrochloric acid. The precipitated solid wasfiltered to give the cyclopropanecarboxylic acid as a white solid.

General Procedure III: Carboxylic Acid Building Block

B. 1-(2,2-Difluoro-benzol-1,31-dioxol-5-yl)-cyclopropanecarboxylic acid

Step a: 2,2-Difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester

A solution of 5-bromo-2,2-difluoro-benzo[1,3]dioxole (11.8 g, 50.0 mmol)and tetrakis(triphenylphosphine)palladium (0) [Pd(PPh₃)₄, 5.78 g, 5.00mmol] in methanol (20 mL) containing acetonitrile (30 mL) andtriethylamine (10 mL) was stirred under a carbon monoxide atmosphere (55PSI) at 75° C. (oil bath temperature) for 15 hours. The cooled reactionmixture was filtered and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography to give crude2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester (11.5 g),which was used directly in the next step.

Step b: (2,2-Difluoro-benzo[1,3]dioxol-5-yl)-methanol

Crude 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid methyl ester(11.5 g) dissolved in 20 mL of anhydrous tetrahydrofuran (THF) wasslowly added to a suspension of lithium aluminum hydride (4.10 g, 106mmol) in anhydrous THF (100 mL) at 0° C. The mixture was then warmed toroom temperature. After being stirred at room temperature for 1 hour,the reaction mixture was cooled to 0° C. and treated with water (4.1 g),followed by sodium hydroxide (10% aqueous solution, 4.1 mL). Theresulting slurry was filtered and washed with THF. The combined filtratewas evaporated to dryness and the residue was purified by silica gelcolumn chromatography to give(2,2-difluoro-benzo[1,3]dioxol-5-yl)-methanol (7.2 g, 38 mmol, 76% overtwo steps) as a colorless oil.

Step c: 5-Chloromethyl-2,2-difluoro-benzo[1,3]dioxole

Thionyl chloride (45 g, 38 mmol) was slowly added to a solution of(2,2-difluoro-benzo[1,3]dioxol-5-yl)-methanol (7.2 g, 38 mmol) indichloromethane (200 mL) at 0° C. The resulting mixture was stirredovernight at room temperature and then evaporated to dryness. Theresidue was partitioned between an aqueous solution of saturated sodiumbicarbonate (100 mL) and dichloromethane (100 mL). The separated aqueouslayer was extracted with dichloromethane (150 mL) and the organic layerwas dried over sodium sulfate, filtered, and evaporated to dryness togive crude 5-chloromethyl-2,2-difluoro-benzo[1,3]dioxole (4.4 g) whichwas used directly in the next step.

Step d: (2,2-Difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile

A mixture of crude 5-chloromethyl-2,2-difluoro-benzo[1,3]dioxole (4.4 g)and sodium cyanide (1.36 g, 27.8 mmol) in dimethylsulfoxide (50 mL) wasstirred at room temperature overnight. The reaction mixture was pouredinto ice and extracted with ethyl acetate (300 mL). The organic layerwas dried over sodium sulfate and evaporated to dryness to give crude(2,2-difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile (3.3 g) which was useddirectly in the next step.

Step e: 1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile

Sodium hydroxide (50% aqueous solution, 10 mL) was slowly added to amixture of crude (2,2-difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile,benzyltriethylammonium chloride (3.00 g, 15.3 mmol), and1-bromo-2-chloroethane (4.9 g, 38 mmol) at 70° C. The mixture wasstirred overnight at 70° C. before the reaction mixture was diluted withwater (30 mL) and extracted with ethyl acetate. The combined organiclayers were dried over sodium sulfate and evaporated to dryness to givecrude 1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile,which was used directly in the next step.

Step f: 1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylicacid

1-(2,2-Difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarbonitrile (crudefrom the last step) was refluxed in 10% aqueous sodium hydroxide (50 mL)for 2.5 hours. The cooled reaction mixture was washed with ether (100mL) and the aqueous phase was acidified to pH 2 with 2M hydrochloricacid. The precipitated solid was filtered to give1-(2,2-difluoro-benzo[1,3]dioxol-5-yl)-cyclopropanecarboxylic acid as awhite solid (0.15 g, 1.6% over four steps). ESI-MS m/z calc. 242.2.found 243.3 (M+1)⁺; ¹H NMR (CDCl₃) δ 7.14-7.04 (m, 2H), 6.98-6.96 (m,1H), 1.74-1.64 (m, 2H), 1.26-1.08 (m, 2H).

C. 2-(4-Chloro-3-methoxyphenyl)acetonitrile

Step a: 1-Chloro-2-methoxy-4-methyl-benzene

To a solution of 2-chloro-5-methyl-phenol (93 g, 0.65 mol) in CH₃CN (700mL) was added CH₃I (111 g, 0.78 mol) and K₂CO₃ (180 g, 1.3 mol). Themixture was stirred at 25° C. overnight. The solid was filtered off andthe filtrate was evaporated under vacuum to give1-chloro-2-methoxy-4-methyl-benzene (90 g, 89%). ¹H NMR (300 MHz, CDCl₃)δ 7.22 (d, J=7.8 Hz, 1H), 6.74-6.69 (m, 2H), 3.88 (s, 3H), 2.33 (s, 3H).

Step b: 4-Bromomethyl-1-chloro-2-methoxy-benzene

To a solution of 1-chloro-2-methoxy-4-methyl-benzene (50 g, 0.32 mol) inCCl₄ (350 mL) was added NBS (57.2 g, 0.32 mol) and AIBN (10 g, 60 mmol).The mixture was heated at reflux for 3 hours. The solvent was evaporatedunder vacuum and the residue was purified by column chromatography onsilica gel (Petroleum Ether/EtOAc=20:1) to give4-bromomethyl-1-chloro-2-methoxy-benzene (69 g, 92%). ¹H NMR (400 MHz,CDCl₃) δ 7.33-7.31 (m, 1H), 6.95-6.91 (m, 2H), 4.46 (s, 2H), 3.92 (s,3H).

Step c: 2-(4-Chloro-3-methoxyphenyl)acetonitrile

To a solution of 4-bromomethyl-1-chloro-2-methoxy-benzene (68.5 g, 0.29mol) in C₂H₅OH (90%, 500 mL) was added NaCN (28.5 g, 0.58 mol). Themixture was stirred at 60° C. overnight. Ethanol was evaporated and theresidue was dissolved in H₂O. The mixture was extracted with ethylacetate (300 mL×3). The combined organic layers were washed with brine,dried over Na₂SO₄ and purified by column chromatography on silica gel(Petroleum Ether/EtOAc 30:1) to give2-(4-chloro-3-methoxyphenyl)acetonitrile (25 g, 48%). ¹H NMR (400 MHz,CDCl₃) δ 7.36 (d, J=8 Hz, 1H), 6.88-6.84 (m, 2H), 3.92 (s, 3H), 3.74 (s,2H). ¹³C NMR (100 MHz, CDCl₃) δ 155.4, 130.8, 129.7, 122.4, 120.7,117.5, 111.5, 56.2, 23.5.

D. (4-Chloro-3-hydroxy-phenyl)-acetonitrile

BBr₃ (16.6 g, 66 mmol) was slowly added to a solution of2-(4-chloro-3-methoxyphenyl)acetonitrile (12 g, 66 mmol) in DCM (120 mL)at −78° C. under N₂. The reaction temperature was slowly increased toroom temperature. The reaction mixture was stirred overnight and thenpoured into ice-water. The organic layer was separated and the aqueouslayer was extracted with DCM (40 mL×3). The combined organic layers werewashed with water, brine, dried over Na₂SO₄, and concentrated undervacuum to give (4-chloro-3-hydroxy-phenyl)-acetonitrile (9.3 g, 85%). ¹HNMR (300 MHz, CDCl₃) δ 7.34 (d, J=8.4 Hz, 1H), 7.02 (d, J=2.1 Hz, 1H),6.87 (dd, J=2.1, 8.4 Hz, 1H), 5.15 (brs, 1H), 3.72 (s, 2H).

E. 1-(3-(Hydroxymethyl)-4-methoxyphenyl)cyclopropanecarboxylic acid

Step a: 1-(4-Methoxy-phenyl)-cyclopropanecarboxylic acid methyl ester

To a solution of 1-(4-methoxy-phenyl)-cyclopropanecarboxylic acid (50.0g, 0.26 mol) in MeOH (500 mL) was added toluene-4-sulfonic acidmonohydrate (2.5 g, 13 mmol) at room temperature. The reaction mixturewas heated at reflux for 20 hours. MeOH was removed by evaporation undervacuum and EtOAc (200 mL) was added. The organic layer was washed withsat. aq. NaHCO₃ (100 mL) and brine, dried over anhydrous Na₂SO₄ andevaporated under vacuum to give1-(4-methoxy-phenyl)-cyclopropanecarboxylic acid methyl ester (53.5 g,99%). ¹H NMR (CDCl₃, 400 MHz) δ 7.25-7.27 (m, 2H), 6.85 (d, J=8.8 Hz,2H), 3.80 (s, 3H), 3.62 (s, 3H), 1.58 (m, 2H), 1.15 (m, 2H).

Step b: 1-(3-Chloromethyl-4-methoxy-phenyl)-cyclopropanecarboxylic acidmethyl ester

To a solution of 1-(4-methoxy-phenyl)-cyclopropanecarboxylic acid methylester (30.0 g, 146 mmol) and MOMCl (29.1 g, 364 mmol) in CS₂ (300 mL)was added TiCl₄ (8.30 g, 43.5 mmol) at 5° C. The reaction mixture washeated at 30° C. for 1 day and poured into ice-water. The mixture wasextracted with CH₂Cl₂ (150 mL×3). The combined organic extracts wereevaporated under vacuum to give crude1-(3-chloromethyl-4-methoxy-phenyl)-cyclopropanecarboxylic acid methylester (38.0 g), which was used in the next step without furtherpurification.

Step c: 1-(3-Hydroxymethyl-4-methoxy-phenyl)-cyclopropanecarboxylic acidmethyl ester

To a suspension of crude1-(3-chloromethyl-4-methoxy-phenyl)-cyclopropanecarboxylic acid methylester (20.0 g) in water (350 mL) was added Bu₄NBr (4.0 g) and Na₂CO₃(90.0 g, 0.85 mol) at room temperature. The reaction mixture was heatedat 65° C. overnight. The resulting solution was acidified with aq. HCl(2 mol/L) and extracted with EtOAc (200 mL×3). The organic layer waswashed with brine, dried over anhydrous Na₂SO₄ and evaporated undervacuum to give crude product, which was purified by column (PetroleumEther/EtOAc 15:1) to give1-(3-hydroxymethyl-4-methoxy-phenyl)-cyclopropanecarboxylic acid methylester (8.0 g, 39%). ¹H NMR (CDCl₃, 400 MHz) δ 7.23-7.26 (m, 2H), 6.83(d, J=8.0 Hz, 1H), 4.67 (s, 2H), 3.86 (s, 3H), 3.62 (s, 3H), 1.58 (q,J=3.6 Hz, 2H), 1.14-1.17 (m, 2H).

Step d:1-[3-(tert-Butyl-dimethyl-silanyloxymethyl)-4-methoxy-phenyl]cyclopropane-carboxylicacid methyl ester

To a solution of1-(3-hydroxymethyl-4-methoxy-phenyl)-cyclopropanecarboxylic acid methylester (8.0 g, 34 mmol) in CH₂Cl₂ (100 mL) were added imidazole (5.8 g,85 mmol) and TBSCl (7.6 g, 51 mmol) at room temperature. The mixture wasstirred overnight at room temperature. The mixture was washed withbrine, dried over anhydrous Na₂SO₄ and evaporated under vacuum to givecrude product, which was purified by column (Petroleum Ether/EtOAc 30:1)to give1-[3-(tert-butyl-dimethyl-silanyloxymethyl)-4-methoxy-phenyl]-cyclopropanecarboxylicacid methyl ester (6.7 g, 56%). ¹H NMR (CDCl₃, 400 MHz) δ 7.44-7.45 (m,1H), 7.19 (dd, J=2.0, 8.4 Hz, 1H), 6.76 (d, J=8.4 Hz, 1H), 4.75 (s, 2H),3.81 (s, 3H), 3.62 (s, 3H), 1.57-1.60 (m, 2H), 1.15-1.18 (m, 2H), 0.96(s, 9H), 0.11 (s, 6H).

Step e: 1-(3-Hydroxymethyl-4-methoxy-phenyl)-cyclopropanecarboxylic acid

To a solution of1-[3-(tert-butyl-dimethyl-silanyloxymethyl)-4-methoxy-phenyl]-cyclopropanecarboxylicacid methyl ester (6.2 g, 18 mmol) in MeOH (75 mL) was added a solutionof LiOH.H₂O (1.50 g, 35.7 mmol) in water (10 mL) at 0° C. The reactionmixture was stirred overnight at 40° C. MeOH was removed by evaporationunder vacuum. AcOH (1 mol/L, 40 mL) and EtOAc (200 mL) were added. Theorganic layer was separated, washed with brine, dried over anhydrousNa₂SO₄ and evaporated under vacuum to provide1-(3-hydroxymethyl-4-methoxy-phenyl)-cyclopropanecarboxylic acid (5.3g).

F. 2-(3-Fluoro-4-methoxyphenyl)acetonitrile

To a suspension of t-BuOK (25.3 g, 0.207 mol) in THF (150 mL) was addeda solution of TosMIC (20.3 g, 0.104 mol) in THF (50 mL) at −78° C. Themixture was stirred for 15 minutes, treated with a solution of3-fluoro-4-methoxy-benzaldehyde (8.00 g, 51.9 mmol) in THF (50 mL)dropwise, and continued to stir for 1.5 hours at −78° C. To the cooledreaction mixture was added methanol (50 mL). The mixture was heated atreflux for 30 minutes. Solvent of the reaction mixture was removed togive a crude product, which was dissolved in water (200 mL). The aqueousphase was extracted with EtOAc (100 mL×3). The combined organic layerswere dried and evaporated under reduced pressure to give crude product,which was purified by column chromatography (Petroleum Ether/EtOAc 10:1)to afford 2-(3-fluoro-4-methoxyphenyl)acetonitrile (5.0 g, 58%). ¹H NMR(400 MHz, CDCl₃) δ 7.02-7.05 (m, 2H), 6.94 (t, J=8.4 Hz, 1H), 3.88 (s,3H), 3.67 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 152.3, 147.5, 123.7,122.5, 117.7, 115.8, 113.8, 56.3, 22.6.

G. 2-(3-Chloro-4-methoxyphenyl)acetonitrile

To a suspension of t-BuOK (4.8 g, 40 mmol) in THF (30 mL) was added asolution of TosMIC (3.9 g, 20 mmol) in THF (10 mL) at −78° C. Themixture was stirred for 10 minutes, treated with a solution of3-chloro-4-methoxy-benzaldehyde (1.65 g, 10 mmol) in THF (10 mL)dropwise, and continued to stir for 1.5 hours at −78° C. To the cooledreaction mixture was added methanol (10 mL). The mixture was heated atreflux for 30 minutes. Solvent of the reaction mixture was removed togive a crude product, which was dissolved in water (20 mL). The aqueousphase was extracted with EtOAc (20 mL×3). The combined organic layerswere dried and evaporated under reduced pressure to give crude product,which was purified by column chromatography (Petroleum Ether/EtOAc 10:1)to afford 2-(3-chloro-4-methoxyphenyl)acetonitrile (1.5 g, 83%). ¹H NMR(400 MHz, CDCl₃) δ 7.33 (d, J=2.4 Hz, 1H), 7.20 (dd, J=2.4, 8.4 Hz, 1H),6.92 (d, J=8.4 Hz, 1H), 3.91 (s, 3H), 3.68 (s, 2H). ¹³C NMR (100 MHz,CDCl₃) δ 154.8, 129.8, 127.3, 123.0, 122.7, 117.60, 112.4, 56.2, 22.4.

H. 1-(3,3-Dimethyl-2,3-dihydrobenzofuran-5-yl)cyclopropanecarboxylicacid

Step a: 1-(4-Hydroxy-phenyl)-cyclopropanecarboxylic acid methyl ester

To a solution of methyl 1-(4-methoxyphenyl)cyclopropanecarboxylate (10.0g, 48.5 mmol) in DCM (80 mL) was added EtSH (16 mL) under ice-waterbath. The mixture was stirred at 0° C. for 20 min before AlCl₃ (19.5 g,0.15 mmol) was added slowly at 0° C. The mixture was stirred at 0° C.for 30 min. The reaction mixture was poured into ice-water, the organiclayer was separated, and the aqueous phase was extracted with DCM (50mL×3). The combined organic layers were washed with H₂O, brine, driedover Na₂SO₄ and evaporated under vacuum to give1-(4-hydroxy-phenyl)-cyclopropanecarboxylic acid methyl ester (8.9 g,95%). ¹H NMR (400 MHz, CDCl₃) δ 7.20-7.17 (m, 2H), 6.75-6.72 (m, 2H),5.56 (s, 1H), 3.63 (s, 3H), 1.60-1.57 (m, 2H), 1.17-1.15 (m, 2H).

Step b: 1-(4-Hydroxy-3,5-diiodo-phenyl)-cyclopropanecarboxylic acidmethyl ester

To a solution of 1-(4-hydroxy-phenyl)-cyclopropanecarboxylic acid methylester (8.9 g, 46 mmol) in CH₃CN (80 mL) was added NIS (15.6 g, 69 mmol).The mixture was stirred at room temperature for 1 hour. The reactionmixture was concentrated and the residue was purified by columnchromatography on silica gel (Petroleum Ether/EtOAc 10:1) to give1-(4-hydroxy-3,5-diiodo-phenyl)-cyclopropanecarboxylic acid methyl ester(3.5 g, 18%). ¹H NMR (400 MHz, CDCl₃) δ 7.65 (s, 2H), 5.71 (s, 1H), 3.63(s, 3H), 1.59-1.56 (m, 2H), 1.15-1.12 (m, 2H).

Step c:1-[3,5-Diiodo-4-(2-methyl-allyloxy)-phenyl]-cyclopropanecarboxylic acidmethyl ester

A mixture of 1-(4-hydroxy-3,5-diiodo-phenyl)-cyclopropanecarboxylic acidmethyl ester (3.2 g, 7.2 mmol), 3-chloro-2-methyl-propene (1.0 g, 11mmol), K₂CO₃ (1.2 g, 8.6 mmol), NaI (0.1 g, 0.7 mmol) in acetone (20 mL)was stirred at 20° C. overnight. The solid was filtered off and thefiltrate was concentrated under vacuum to give1-[3,5-d]iodo-4-(2-methyl-allyloxy)-phenyl]-cyclopropane-carboxylic acidmethyl ester (3.5 g, 97%). ¹H NMR (300 MHz, CDCl₃) δ 7.75 (s, 2H), 5.26(s, 1H), 5.06 (s, 1H), 4.38 (s, 2H), 3.65 (s, 3H), 1.98 (s, 3H),1.62-1.58 (m, 2H), 1.18-1.15 (m, 2H).

Step d:1-(3,3-Dimethyl-2,3-dihydro-benzofuran-5-yl)-cyclopropanecarboxylic acidmethyl ester

To a solution of1-[3,5-d]iodo-4-(2-methyl-allyloxy)-phenyl]-cyclopropane-carboxylic acidmethyl ester (3.5 g, 7.0 mmol) in toluene (15 mL) was added Bu₃SnH (2.4g, 8.4 mmol) and AIBN (0.1 g, 0.7 mmol). The mixture was heated atreflux overnight. The reaction mixture was concentrated under vacuum andthe residue was purified by column chromatography on silica gel(Petroleum Ether/EtOAc 20:1) to give1-(3,3-dimethyl-2,3-dihydro-benzofuran-5-yl)-cyclopropanecarboxylic acidmethyl ester (1.05 g, 62%). ¹H NMR (400 MHz, CDCl₃) δ 7.10-7.07 (m, 2H),6.71 (d, J=8 Hz, 1H), 4.23 (s, 2H), 3.62 (s, 3H), 1.58-1.54 (m, 2H),1.34 (s, 6H), 1.17-1.12 (m, 2H).

Step e:1-(3,3-Dimethyl-2,3-dihydrobenzofuran-5-yl)cyclopropanecarboxylic acid

To a solution of1-(3,3-dimethyl-2,3-dihydro-benzofuran-5-yl)-cyclopropanecarboxylic acidmethyl ester (1 g, 4 mmol) in MeOH (10 mL) was added LiOH (0.40 g, 9.5mmol). The mixture was stirred at 40° C. overnight. HCl (10%) was addedslowly to adjust the pH to 5. The resulting mixture was extracted withethyl acetate (10 mL×3). The extracts were washed with brine and driedover Na₂SO₄. The solvent was removed under vacuum and the crude productwas purified by preparative HPLC to give1-(3,3-dimethyl-2,3-dihydrobenzofuran-5-yl)cyclopropanecarboxylic acid(0.37 g, 41%). ¹H NMR (400 MHz, CDCl₃) δ 7.11-7.07 (m, 2H), 6.71 (d, J=8Hz, 1H), 4.23 (s, 2H), 1.66-1.63 (m, 2H), 1.32 (s, 6H), 1.26-1.23 (m,2H).

I. 2-(7-Methoxybenzo[d][1,3]-dioxol-5-yl)acetonitrile

Step a: 3,4-Dihydroxy-5-methoxybenzoate

To a solution of 3,4,5-trihydroxy-benzoic acid methyl ester (50 g, 0.27mol) and Na₂B₄O₇ (50 g) in water (1000 mL) was added Me₂SO₄ (120 mL) andaqueous NaOH solution (25%, 200 mL) successively at room temperature.The mixture was stirred at room temperature for 6 h before it was cooledto 0° C. The mixture was acidified to pH ˜2 by adding conc. H₂SO₄ andthen filtered. The filtrate was extracted with EtOAc (500 mL×3). Thecombined organic layers were dried over anhydrous Na₂SO₄ and evaporatedunder reduced pressure to give methyl 3,4-dihydroxy-5-methoxybenzoate(15.3 g 47%), which was used in the next step without furtherpurification.

Step b: Methyl 7-methoxybenzo[d][1,3]dioxole-5-carboxylate

To a solution of methyl 3,4-dihydroxy-5-methoxybenzoate (15.3 g, 0.078mol) in acetone (500 mL) was added CH₂BrCl (34.4 g, 0.27 mol) and K₂CO₃(75 g, 0.54 mol) at 80° C. The resulting mixture was heated at refluxfor 4 h. The mixture was cooled to room temperature and solid K₂CO₃ wasfiltered off. The filtrate was concentrated under reduced pressure, andthe residue was dissolved in EtOAc (100 mL). The organic layer waswashed with water, dried over anhydrous Na₂SO₄, and evaporated underreduced pressure to give the crude product, which was purified by columnchromatography on silica gel (Petroleum Ether/Ethyl Acetate=10:1) toafford methyl 7-methoxybenzo[d][1,3]dioxole-5-carboxylate (12.6 g, 80%).¹H NMR (400 MHz, CDCl₃) δ 7.32 (s, 1H), 7.21 (s, 1H), 6.05 (s, 2H), 3.93(s, 3H), 3.88 (s, 3H).

Step c: (7-Methoxybenzo[d][1,3]dioxol-5-yl)methanol

To a solution of methyl 7-methoxybenzo[d][1,3]dioxole-5-carboxylate(13.9 g, 0.040 mol) in THF (100 mL) was added LiAlH₄ (3.1 g, 0.080 mol)in portions at room temperature. The mixture was stirred for 3 h at roomtemperature. The reaction mixture was cooled to 0° C. and treated withwater (3.1 g) and NaOH (10%, 3.1 mL) successively. The slurry wasfiltered off and washed with THF. The combined filtrates were evaporatedunder reduced pressure to give(7-methoxy-benzo[d][1,3]dioxol-5-yl)methanol (7.2 g, 52%). ¹H NMR (400MHz, CDCl₃) δ 6.55 (s, 1H), 6.54 (s, 1H), 5.96 (s, 2H), 4.57 (s, 2H),3.90 (s, 3H).

Step d: 6-(Chloromethyl)-4-methoxybenzo[d][1,3]dioxole

To a solution of SOCl₂ (150 mL) was added(7-methoxybenzo[d][1,3]dioxol-5-yl)methanol (9.0 g, 54 mmol) in portionsat 0° C. The mixture was stirred for 0.5 h. The excess SOCl₂ wasevaporated under reduced pressure to give the crude product, which wasbasified with sat. aq. NaHCO₃ to pH ˜7. The aqueous phase was extractedwith EtOAc (100 mL×3). The combined organic layers were dried overanhydrous Na₂SO₄ and evaporated to give6-(chloromethyl)-4-methoxybenzo[d][1,3]dioxole (10.2 g 94%), which wasused in the next step without further purification. ¹H NMR (400 MHz,CDCl₃) δ 6.58 (s, 1H), 6.57 (s, 1H), 5.98 (s, 2H), 4.51 (s, 2H), 3.90(s, 3H).

Step e: 2-(7-Methoxybenzo[d][1,3]dioxol-5-yl)acetonitrile

To a solution of 6-(chloromethyl)-4-methoxybenzo[d][1,3]dioxole (10.2 g,40 mmol) in DMSO (100 mL) was added NaCN (2.43 g, 50 mmol) at roomtemperature. The mixture was stirred for 3 h and poured into water (500mL). The aqueous phase was extracted with EtOAc (100 mL×3). The combinedorganic layers were dried over anhydrous Na₂SO₄ and evaporated to givethe crude product, which was washed with ether to afford2-(7-methoxybenzo[d][1,3]dioxol-5-yl)acetonitrile (4.6 g, 45%). ¹H NMR(400 MHz, CDCl₃) δ 6.49 (s, 2H), 5.98 (s, 2H), 3.91 (s, 3H), 3.65 (s,2H). ¹³C NMR (400 MHz, CDCl₃) δ 148.9, 143.4, 134.6, 123.4, 117.3,107.2, 101.8, 101.3, 56.3, 23.1.

J. 1-(Benzofuran-5-yl)cyclopropanecarboxylic acid

Step a: 1-[4-(2,2-Diethoxy-ethoxy)-phenyl]-cyclopropanecarboxylic acid

To a stirred solution of 1-(4-hydroxy-phenyl)-cyclopropanecarboxylicacid methyl ester (15.0 g, 84.3 mmol) in DMF (50 mL) was added sodiumhydride (6.7 g, 170 mmol, 60% in mineral oil) at 0° C. After hydrogenevolution ceased, 2-bromo-1,1-diethoxy-ethane (16.5 g, 84.3 mmol) wasadded dropwise to the reaction mixture. The reaction was stirred at 160°C. for 15 hours. The reaction mixture was poured onto ice (100 g) andextracted with CH₂Cl₂. The combined organics were dried over Na₂SO₄. Thesolvent was evaporated under vacuum to give crude1-[4-(2,2-diethoxy-ethoxy)-phenyl]-cyclopropanecarboxylic acid (10 g),which was used directly in the next step without purification.

Step b: 1-Benzofuran-5-yl-cyclopropanecarboxylic acid

To a suspension of crude1-[4-(2,2-diethoxy-ethoxy)-phenyl]-cyclopropanecarboxylic acid (20 g,˜65 mmol) in xylene (100 mL) was added PPA (22.2 g, 64.9 mmol) at roomtemperature. The mixture was heated at reflux (140° C.) for 1 hourbefore it was cooled to room temperature and decanted from the PPA. Thesolvent was evaporated under vacuum to obtain the crude product, whichwas purified by preparative HPLC to provide1-(benzofuran-5-yl)cyclopropanecarboxylic acid (1.5 g, 5%). ¹H NMR (400MHz, DMSO-d₆) δ 12.25 (br s, 1H), 7.95 (d, J=2.8 Hz, 1H), 7.56 (d, J=2.0Hz, 1H), 7.47 (d, J=11.6 Hz, 1H), 7.25 (dd, J=2.4, 11.2 Hz, 1H), 6.89(d, J=1.6 Hz, 1H), 1.47-1.44 (m, 2H), 1.17-1.14 (m, 2H).

K. 1-(2,3-Dihydrobenzofuran-5-yl)cyclopropanecarboxylic acid

To a solution of 1-(benzofuran-5-yl)cyclopropanecarboxylic acid (700 mg,3.47 mmol) in MeOH (10 mL) was added PtO₂ (140 mg, 20%) at roomtemperature. The stirred reaction mixture was hydrogenated underhydrogen (1 atm) at 10° C. for 3 days. The reaction mixture wasfiltered. The solvent was evaporated under vacuum to afford the crudeproduct, which was purified by preparative HPLC to give1-(2,3-dihydrobenzofuran-5-yl)cyclopropanecarboxylic acid (330 mg, 47%).¹H NMR (400 MHz, CDCl₃) δ 7.20 (s, 1H), 7.10 (d, J=10.8 Hz, 1H), 6.73(d, J=11.2 Hz, 1H), 4.57 (t, J=11.6 Hz, 2H), 3.20 (t, J=11.6 Hz, 2H),1.67-1.63 (m, 2H), 1.25-1.21 (m, 2H).

L. 2-(2,2-Dimethylbenzo[d][1,3]-dioxol-5-yl)acetonitrile

Step a: (3,4-Dihydroxy-phenyl)-acetonitrile

To a solution of benzo[1,3]dioxol-5-yl-acetonitrile (0.50 g, 3.1 mmol)in CH₂Cl₂ (15 mL) was added dropwise BBr₃ (0.78 g, 3.1 mmol) at −78° C.under N₂. The mixture was slowly warmed to room temperature and stirredovernight. H₂O (10 mL) was added to quench the reaction and the CH₂Cl₂layer was separated. The aqueous phase was extracted with CH₂Cl₂ (2×7mL). The combined organics were washed with brine, dried over Na₂SO₄ andpurified by column chromatography on silica gel (Petroleum Ether/EtOAc5:1) to give (3,4-dihydroxy-phenyl)-acetonitrile (0.25 g, 54%) as awhite solid. ¹H NMR (DMSO-d₆, 400 MHz) δ 9.07 (s, 1H), 8.95 (s, 1H),6.68-6.70 (m, 2H), 6.55 (dd, J=8.0, 2.0 Hz, 1H), 3.32 (s, 2H).

Step b: 2-(2,2-Dimethylbenzo[d][1,3]dioxol-5-yl)acetonitrile

To a solution of (3,4-dihydroxy-phenyl)-acetonitrile (0.2 g, 1.3 mmol)in toluene (4 mL) was added 2,2-dimethoxy-propane (0.28 g, 2.6 mmol) andTsOH (0.010 g, 0.065 mmol). The mixture was heated at reflux overnight.The reaction mixture was evaporated to remove the solvent and theresidue was dissolved in ethyl acetate. The organic layer was washedwith NaHCO₃ solution, H₂O, brine, and dried over Na₂SO₄. The solvent wasevaporated under reduced pressure to give a residue, which was purifiedby column chromatography on silica gel (Petroleum Ether/EtOAc 10:1) togive 2-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)acetonitrile (40 mg, 20%).¹H NMR (CDCl₃, 400 MHz) δ 6.68-6.71 (m, 3H), 3.64 (s, 2H), 1.67 (s, 6H).

M. 2-(3-(Benzyloxy)-4-chlorophenyl)acetonitrile

Step a: (4-Chloro-3-hydroxy-phenyl)acetonitrile

BBr₃ (16.6 g, 66 mmol) was slowly added to a solution of2-(4-chloro-3-methoxyphenyl)acetonitrile (12 g, 66 mmol) in DCM (120 mL)at −78° C. under N₂. The reaction temperature was slowly increased toroom temperature. The reaction mixture was stirred overnight and thenpoured into ice and water. The organic layer was separated, and theaqueous layer was extracted with DCM (40 mL×3). The combined organiclayers were washed with water, brine, dried over Na₂SO₄, andconcentrated under vacuum to give(4-chloro-3-hydroxy-phenyl)-acetonitrile (9.3 g, 85%). ¹H NMR (300 MHz,CDCl₃) δ 7.34 (d, J=8.4 Hz, 1H), 7.02 (d, J=2.1 Hz, 1H), 6.87 (dd,J=2.1, 8.4 Hz, 1H), 5.15 (brs, 1H), 3.72 (s, 2H).

Step b: 2-(3-(Benzyloxy)-4-chlorophenyl)acetonitrile

To a solution of (4-chloro-3-hydroxy-phenyl)acetonitrile (6.2 g, 37mmol) in CH₃CN (80 mL) was added K₂CO₃ (10.2 g, 74 mmol) and BnBr (7.6g, 44 mmol). The mixture was stirred at room temperature overnight. Thesolids were filtered off and the filtrate was evaporated under vacuum.The residue was purified by column chromatography on silica gel(Petroleum Ether/Ethyl Acetate 50:1) to give2-(3-(benzyloxy)-4-chlorophenyl)acetonitrile (5.6 g, 60%). ¹H NMR (400MHz, CDCl₃) δ 7.48-7.32 (m, 6H), 6.94 (d, J=2 Hz, 2H), 6.86 (dd, J=2.0,8.4 Hz, 1H), 5.18 (s, 2H), 3.71 (s, 2H).

N. 2-(Quinoxalin-6-yl)acetonitrile

Step a: 6-Methylquinoxaline

To a solution of 4-methylbenzene-1,2-diamine (50.0 g, 0.41 mol) inisopropanol (300 mL) was added a solution of glyoxal (40% in water, 65.3g, 0.45 mol) at room temperature. The reaction mixture was heated at 80°C. for 2 hours and evaporated under vacuum to give 6-methylquinoxaline(55 g, 93%), which was used directly in the next step. ¹H NMR (300 MHz,CDCl₃) δ 8.77 (dd, J=1.5, 7.2 Hz, 2H), 7.99 (d, J=8.7 Hz, 1H), 7.87 (s,1H), 7.60 (dd, J=1.5, 8.4 Hz, 1H), 2.59 (s, 3H).

Step b: 6-Bromomethylquinoxaline

To a solution of 6-methylquinoxaline (10.0 g, 69.4 mmol) in CCl₄ (80 mL)was added NBS (13.5 g, 76.3 mmol) and benzoyl peroxide (BP, 1.7 g, 6.9mmol) at room temperature. The mixture was heated at reflux for 2 hours.After cooling, the mixture was evaporated under vacuum to give a yellowsolid, which was extracted with Petroleum Ether (50 mL×5). The extractswere concentrated under vacuum. The organics were combined andconcentrated to give crude 6-bromomethylquinoxaline (12.0 g), which wasused directly in the next step. ¹H NMR (300 MHz, CDCl₃) δ 8.85-8.87 (m,2H), 8.10-8.13 (m, 2H), 7.82 (dd, J=2.1, 8.7 Hz, 1H), 4.70 (s, 2H).

Step c: 2-(Quinoxalin-6-yl)acetonitrile

To a solution of crude 6-bromomethylquinoxaline (36.0 g) in 95% ethanol(200 mL) was added NaCN (30.9 g, 0.63 mol) at room temperature. Themixture was heated at 50° C. for 3 hours and then concentrated undervacuum. Water (100 mL) and ethyl acetate (100 mL) were added. Theorganic layer was separated and the aqueous layer was extracted withethyl acetate. The combined organics were washed with brine, dried overNa₂SO₄ and concentrated under vacuum. The residue was purified by silicagel column (Petroleum Ether/EtOAc 10:1) to give2-(quinoxalin-6-yl)acetonitrile (7.9 g, 23% over two steps). ¹H NMR (300MHz, CDCl₃) δ 8.88-8.90 (m, 2H), 8.12-8.18 (m, 2H), 7.74 (dd, J=2.1, 8.7Hz, 1H), 4.02 (s, 2H). MS (ESI) m/z (M+H)⁺170.0.

O. 2-(Quinolin-6-yl)acetonitrile

Step a: 6-Bromomethylquinoline

To a solution of 6-methylquinoline (2.15 g, 15.0 mmol) in CCl₄ (30 mL)was added NBS (2.92 g, 16.5 mmol) and benzoyl peroxide (BP, 0.36 g, 1.5mmol) at room temperature. The mixture was heated at reflux for 2 hours.After cooling, the mixture was evaporated under vacuum to give a yellowsolid, which was extracted with Petroleum Ether (30 mL×5). The extractswere concentrated under vacuum to give crude 6-bromomethylquinoline (1.8g), which was used directly in the next step.

Step b: 2-(Quinolin-6-yl)acetonitrile

To a solution of crude 6-bromomethylquinoline (1.8 g) in 95% ethanol (30mL) was added NaCN (2.0 g, 40.8 mmol) at room temperature. The mixturewas heated at 50° C. for 3 hours and then concentrated under vacuum.Water (50 mL) and ethyl acetate (50 mL) were added. The organic layerwas separated and the aqueous layer was extracted with ethyl acetate.The combined organics were washed with brine, dried over Na₂SO₄ andconcentrated under vacuum. The combined crude product was purified bycolumn (Petroleum Ether/EtOAc 5:1) to give 2-(quinolin-6-yl)acetonitrile(0.25 g, 8% over two steps). ¹H NMR (300 MHz, CDCl₃) δ 8.95 (dd, J=1.5,4.2 Hz, 1H), 8.12-8.19 (m, 2H), 7.85 (s, 1H), 7.62 (dd, J=2.1, 8.7 Hz,1H), 7.46 (q, J=4.2 Hz, 1H), 3.96 (s, 2H). MS (ESI) m/e (M+H)⁺169.0.

P. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)acetonitrile

Step a: 2,3-Dihydro-benzo[1,4]dioxine-6-carboxylic acid ethyl ester

To a suspension of Cs₂CO₃ (270 g, 1.49 mol) in DMF (1000 mL) were added3,4-dihydroxybenzoic acid ethyl ester (54.6 g, 0.3 mol) and1,2-dibromoethane (54.3 g, 0.29 mol) at room temperature. The resultingmixture was stirred at 80° C. overnight and then poured into ice-water.The mixture was extracted with EtOAc (200 mL×3). The combined organiclayers were washed with water (200 mL×3) and brine (100 mL), dried overNa₂SO₄ and concentrated to dryness. The residue was purified by column(Petroleum Ether/Ethyl Acetate 50:1) on silica gel to obtain2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid ethyl ester (18 g, 29%).¹H NMR (300 MHz, CDCl₃) δ 7.53 (dd, J=1.8, 7.2 Hz, 2H), 6.84-6.87 (m,1H), 4.22-4.34 (m, 6H), 1.35 (t, J=7.2 Hz, 3H).

Step b: (2,3-Dihydro-benzo[1,4]dioxin-6-yl)-methanol

To a suspension of LAH (2.8 g, 74 mmol) in THF (20 mL) was addeddropwise a solution of 2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acidethyl ester (15 g, 72 mmol) in THF (10 mL) at 0° C. under N₂. Themixture was stirred at room temperature for 1 h and then quenchedcarefully with addition of water (2.8 mL) and NaOH (10%, 28 mL) withcooling. The precipitated solid was filtered off and the filtrate wasevaporated to dryness to obtain(2,3-dihydro-benzo[1,4]dioxin-6-yl)-methanol (10.6 g). ¹H NMR (300 MHz,DMSO-d₆) δ 6.73-6.78 (m, 3H), 5.02 (t, J=5.7 Hz, 1H), 4.34 (d, J=6.0 Hz,2H), 4.17-4.20 (m, 4H).

Step c: 6-Chloromethyl-2,3-dihydro-benzo[1,4]dioxine

A mixture of (2,3-dihydro-benzo[1,4]dioxin-6-yl)methanol (10.6 g) inSOCl₂ (10 mL) was stirred at room temperature for 10 min and then pouredinto ice-water. The organic layer was separated and the aqueous phasewas extracted with dichloromethane (50 mL×3). The combined organiclayers were washed with NaHCO₃ (sat solution), water and brine, driedover Na₂SO₄ and concentrated to dryness to obtain6-chloromethyl-2,3-dihydro-benzo[1,4]dioxine (12 g, 88% over two steps),which was used directly in next step.

Step d: 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)acetonitrile

A mixture of 6-chloromethyl-2,3-dihydro-benzo[1,4]dioxine (12.5 g, 67.7mmol) and NaCN (4.30 g, 87.8 mmol) in DMSO (50 mL) was stirred at rt for1 h. The mixture was poured into water (150 mL) and then extracted withdichloromethane (50 mL×4). The combined organic layers were washed withwater (50 mL×2) and brine (50 mL), dried over Na₂SO₄ and concentrated todryness. The residue was purified by column (Petroleum Ether/EthylAcetate 50:1) on silica gel to obtain2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acetonitrile as a yellow oil(10.2 g, 86%). ¹H-NMR (300 MHz, CDCl₃) δ 6.78-6.86 (m, 3H), 4.25 (s,4H), 3.63 (s, 2H).

Q. 2-(2,2,4,4-Tetrafluoro-4H-benzo[d][1,3]dioxin-6-yl)acetonitrile

Step a: 2,2,4,4-Tetrafluoro-4H-benzo[1,3]dioxine-6-carboxylic acidmethyl ester

A suspension of 6-bromo-2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxine (4.75g, 16.6 mmol) and Pd(PPh₃)₄ (950 mg, 8.23 mmol) in MeOH (20 mL), MeCN(30 mL) and Et₃N (10 mL) was stirred under carbon monoxide atmosphere(55 psi) at 75° C. (oil bath temperature) overnight. The cooled reactionmixture was filtered and the filtrate was concentrated. The residue waspurified by silica gel column (Petroleum Ether) to give2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxine-6-carboxylic acid methyl ester(3.75 g, 85%). ¹H NMR (CDCl₃, 300 MHz) δ 8.34 (s, 1H), 8.26 (dd, J=2.1,8.7 Hz, 1H), 7.22 (d, J=8.7 Hz, 1H), 3.96 (s, 3H).

Step b: (2,2,4,4-Tetrafluoro-4H-benzo[1,3]dioxin-6-yl)methanol

To a suspension of LAH (2.14 g, 56.4 mmol) in dry THF (200 mL) was addeddropwise a solution of2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxine-6-carboxylic acid methyl ester(7.50 g, 28.2 mmol) in dry THF (50 mL) at 0° C. After being stirred at0° C. for 1 h, the reaction mixture was treated with water (2.14 g) and10% NaOH (2.14 mL). The slurry was filtered and washed with THF. Thecombined filtrates were evaporated to dryness to give the crude(2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxin-6-yl)-methanol (6.5 g), whichwas used directly in the next step. ¹H NMR (CDCl₃, 300 MHz) δ 7.64 (s,1H), 7.57-7.60 (m, 1H), 7.58 (d, J=8.7 Hz, 1H), 4.75 (s, 2H).

Step c: 6-Chloromethyl-2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxine

A mixture of (2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxin-6-yl)-methanol(6.5 g) in thionyl chloride (75 mL) was heated at reflux overnight. Theresulting mixture was concentrated under vacuum. The residue wasbasified with aqueous saturated NaHCO₃. The aqueous layer was extractedwith dichloromethane (50 mL×3). The combined organic layers were driedover Na₂SO₄, filtrated, and concentrated under reduced pressure to give6-chloromethyl-2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxine (6.2 g), whichwas used directly in the next step. ¹H NMR (CDCl₃, 300 MHz) δ 7.65 (s,1H), 7.61 (dd, J=2.1, 8.7 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 4.60 (s, 2H).

Step d: (2,2,4,4-Tetrafluoro-4H-benzo[1,3]dioxin-6-yl)-acetonitrile

A mixture of 6-chloromethyl-2,2,4,4-tetrafluoro-4H-benzo[1,3]dioxine(6.2 g) and NaCN (2.07 g, 42.3 mmol) in DMSO (50 mL) was stirred at roomtemperature for 2 h. The reaction mixture was poured into ice andextracted with EtOAc (50 mL×3). The combined organic layers were driedover anhydrous Na₂SO₄, and evaporated to give a crude product, which waspurified by silica gel column (Petroleum Ether/EtOAc 10:1) to give(2,2-difluoro-benzo[1,3]dioxol-5-yl)-acetonitrile (4.5 g, 68% over 3steps). ¹H NMR (CDCl₃, 300 MHz) δ 7.57-7.60 (m, 2H), 7.20 (d, J=8.7 Hz,1H), 3.82 (s, 2H).

R. 2-(4H-Benzo[d][1,3]dioxin-7-yl)acetonitrile

Step a: (3-Hydroxyphenyl)acetonitrile

To a solution of (3-methoxyphenyl)acetonitrile (150 g, 1.03 mol) inCH₂Cl₂ (1000 mL) was added BBr₃ (774 g, 3.09 mol) dropwise at −70° C.The mixture was stirred and warmed to room temperature slowly. Water(300 mL) was added at 0° C. The resulting mixture was extracted withCH₂Cl₂. The combined organic layers were dried over anhydrous Na₂SO₄,filtered, and evaporated under vacuum. The crude residue was purified bycolumn (Petroleum Ether/EtOAc 10:1) to give(3-hydroxyphenyl)acetonitrile (75.0 g, 55%). ¹H NMR (CDCl₃, 300 MHz) δ7.18-7.24 (m, 1H), 6.79-6.84 (m, 3H), 3.69 (s, 2H).

Step b: 2-(4H-Benzo[d][1,3]dioxin-7-yl)acetonitrile

To a solution of (3-hydroxyphenyl)acetonitrile (75.0 g, 0.56 mol) intoluene (750 mL) was added paraformaldehyde (84.0 g, 2.80 mol) andtoluene-4-sulfonic acid monohydrate (10.7 g, 56.0 mmol) at roomtemperature. The reaction mixture was heated at reflux for 40 minutes.Toluene was removed by evaporation. Water (150 mL) and ethyl acetate(150 mL) were added. The organic layer was separated and the aqueouslayer was extracted with ethyl acetate. The combined organics werewashed with brine, dried over anhydrous Na₂SO₄ and evaporated undervacuum. The residue was separated by preparative HPLC to give2-(4H-benzo[d][1,3]dioxin-7-yl)acetonitrile (4.7 g, 5%). ¹H NMR (300MHz, CDCl₃) δ 6.85-6.98 (m, 3H), 5.25 (d, J=3.0 Hz, 2H), 4.89 (s, 2H),3.69 (s, 2H).

S. 2-(4H-Benzo[d][1,3]dioxin-6-yl)acetonitrile

To a solution of (4-hydroxyphenyl)acetonitrile (17.3 g, 0.13 mol) intoluene (350 mL) were added paraformaldehyde (39.0 g, 0.43 mmol) andtoluene-4-sulfonic acid monohydrate (2.5 g, 13 mmol) at roomtemperature. The reaction mixture was heated at reflux for 1 hour.Toluene was removed by evaporation. Water (150 mL) and ethyl acetate(150 mL) were added. The organic layer was separated and the aqueouslayer was extracted with ethyl acetate. The combined organics werewashed with brine, dried over Na₂SO₄ and evaporated under vacuum. Theresidue was separated by preparative HPLC to give2-(4H-benzo[d][1,3]dioxin-6-yl)acetonitrile (7.35 g, 32%). ¹H NMR (400MHz, CDCl₃) δ 7.07-7.11 (m, 1H), 6.95-6.95 (m, 1H), 6.88 (d, J=11.6 Hz,1H), 5.24 (s, 2H), 4.89 (s, 2H), 3.67 (s, 2H).

T. 2-(3-(Benzyloxy)-4-methoxyphenyl)acetonitrile

To a suspension of t-BuOK (20.15 g, 0.165 mol) in THF (250 mL) was addeda solution of TosMIC (16.1 g, 82.6 mmol) in THF (100 mL) at −78° C. Themixture was stirred for 15 minutes, treated with a solution of3-benzyloxy-4-methoxy-benzaldehyde (10.0 g, 51.9 mmol) in THF (50 mL)dropwise, and continued to stir for 1.5 hours at −78° C. To the cooledreaction mixture was added methanol (50 mL). The mixture was heated atreflux for 30 minutes. Solvent of the reaction mixture was removed togive a crude product, which was dissolved in water (300 mL). The aqueousphase was extracted with EtOAc (100 mL×3). The combined organic layerswere dried and evaporated under reduced pressure to give crude product,which was purified by column chromatography (Petroleum Ether/EtOAc 10:1)to afford 2-(3-(Benzyloxy)-4-methoxyphenyl)acetonitril (5.0 g, 48%). ¹HNMR (300 MHz, CDCl₃) δ 7.48-7.33 (m, 5H), 6.89-6.86 (m, 3H), 5.17 (s,2H), 3.90 (s, 3H), 3.66 (s, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 149.6, 148.6,136.8, 128.8, 128.8, 128.2, 127.5, 127.5, 122.1, 120.9, 118.2, 113.8,112.2, 71.2, 56.2, 23.3.

The following Table 2 contains a list of carboxylic acid building blocksthat were commercially available, or prepared by one of the methodsdescribed above:

TABLE 2 Carboxylic acid building blocks. Compound Name A-11-benzo[1,3]dioxol-5-ylcyclopropane-1-carboxylic acid A-21-(2,2-difluorobenzo[1,3]dioxol-5-yl)cyclopropane-1- carboxylic acid A-31-(3,4-dimethoxyphenyl)cyclopropane-1-carboxylic acid A-41-(3-methoxyphenyl)cyclopropane-1-carboxylic acid A-51-(2-methoxyphenyl)cyclopropane-1-carboxylic acid A-61-[4-(trifluoromethoxy)phenyl]cyclopropane-1-carboxylic acid A-8tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-carboxylic acid A-91-phenylcyclopropane-1-carboxylic acid A-101-(4-methoxyphenyl)cyclopropane-1-carboxylic acid A-111-(4-chlorophenyl)cyclopropane-1-carboxylic acid A-131-phenylcyclopentanecarboxylic acid A-14 1-phenylcyclohexanecarboxylicacid A-15 1-(4-methoxyphenyl)cyclopentanecarboxylic acid A-161-(4-methoxyphenyl)cyclohexanecarboxylic acid A-171-(4-chlorophenyl)cyclohexanecarboxylic acid A-181-(2,3-dihydrobenzo[b][1,4]dioxin-6- yl)cyclopropanecarboxylic acid A-191-(4H-benzo[d][1,3]dioxin-7-yl)cyclopropanecarboxylic acid A-201-(2,2,4,4-tetrafluoro-4H-benzo[d][1,3]dioxin-6-yl)cyclopropanecarboxylic acid A-211-(4H-benzo[d][1,3]dioxin-6-yl)cyclopropanecarboxylic acid A-221-(quinoxalin-6-yl)cyclopropanecarboxylic acid A-231-(quinolin-6-yl)cyclopropanecarboxylic acid A-241-(4-chlorophenyl)cyclopentanecarboxylic acid A-251-(benzofuran-5-yl)cyclopropanecarboxylic acid A-261-(4-chloro-3-methoxyphenyl)cyclopropanecarboxylic acid A-271-(3-(hydroxymethyl)-4- methoxyphenyl)cyclopropanecarboxylic acid A-281-(2,3-dihydrobenzofuran-5-yl)cyclopropanecarboxylic acid A-291-(3-fluoro-4-methoxyphenyl)cyclopropanecarboxylic acid A-301-(3-chloro-4-methoxyphenyl)cyclopropanecarboxylic acid A-311-(3-hydroxy-4-methoxyphenyl)cyclopropanecarboxylic acid A-321-(4-hydroxy-3-methoxyphenyl)cyclopropanecarboxylic acid A-331-(2,2-dimethylbenzo[d][1,3]dioxol-5- yl)cyclopropanecarboxylic acidA-34 1-(3,3-dimethyl-2,3-dihydrobenzofuran-5- yl)cyclopropanecarboxylicacid A-35 1-(7-methoxybenzo[d][1,3]dioxol-5- yl)cyclopropanecarboxylicacid A-36 1-(4-chloro-3-hydroxyphenyl)cyclopropanecarboxylic acid A-371-(4-methoxy-3-methylphenyl)cyclopropanecarboxylic acid A-381-(3-(benzyloxy)-4-chlorophenyl)cyclopropanecarboxylic acid A-451-(4-methoxy-3- (methoxymethyl)phenyl)cyclopropanecarboxylic acid

U. 6-Chloro-5-methylpyridin-2-amine

Step a: 2,2-Dimethyl-N-(5-methyl-pyridin-2-yl)-propionamide

To a stirred solution of 5-methylpyridin-2-amine (200 g, 1.85 mol) inanhydrous CH₂Cl₂ (1000 mL) was added dropwise a solution of Et₃N (513mL, 3.70 mol) and 2,2-dimethyl-propionyl chloride (274 mL, 2.22 mol) at0° C. under N₂. The ice bath was removed and stirring was continued atroom temperature for 2 hours. The reaction was poured into ice (2000 g).The organic layer was separated and the remaining aqueous layer wasextracted with CH₂Cl₂ (3×). The combined organics were dried over Na₂SO₄and evaporated to afford2,2-dimethyl-N-(5-methyl-pyridin-2-yl)-propionamide (350 g), which wasused in the next step without further purification. ¹H NMR (400 MHz,CDCl₃) δ 8.12 (d, J=8.4 Hz, 1H), 8.06 (d, J=1.2 Hz, 1H), 7.96 (s, 1H),7.49 (dd, J=1.6, 8.4 Hz, 1H), 2.27 (s, 1H), 1.30 (s, 9H).

Step b: 2,2-Dimethyl-N-(5-methyl-1-oxy-pyridin-2-yl)-propionamide

To a stirred solution of2,2-dimethyl-N-(5-methyl-pyridin-2-yl)-propionamide (100 g, 0.52 mol) inAcOH (500 mL) was added drop-wise 30% H₂O₂ (80 mL, 2.6 mol) at roomtemperature. The mixture was stirred at 80° C. for 12 hours. Thereaction mixture was evaporated under vacuum to obtain2,2-dimethyl-N-(5-methyl-1-oxy-pyridin-2-yl)-propionamide (80 g, 85%purity). ¹H NMR (400 MHz, CDCl₃) δ 10.26 (br s, 1H), 8.33 (d, J=8.4 Hz,1H), 8.12 (s, 1H), 7.17 (dd, J=0.8, 8.8 Hz, 1H), 2.28 (s, 1H), 1.34 (s,9H).

Step c: N-(6-Chloro-5-methyl-pyridin-2-yl)-2,2-dimethyl-propionamide

To a stirred solution of2,2-dimethyl-N-(5-methyl-1-oxy-pyridin-2-yl)-propionamide (10 g, 48mmol) in anhydrous CH₂Cl₂ (50 mL) was added Et₃N (60 mL, 240 mmol) atroom temperature. After being stirred for 30 min, POCl₃ (20 mL) wasadded drop-wise to the reaction mixture. The reaction was stirred at 50°C. for 15 hours. The reaction mixture was poured into ice (200 g). Theorganic layer was separated and the remaining aqueous layer wasextracted with CH₂Cl₂ (3×). The combined organics were dried overNa₂SO₄. The solvent was evaporated under vacuum to obtain the crudeproduct, which was purified by chromatography (Petroleum Ether/EtOAc100:1) to provideN-(6-chloro-5-methyl-pyridin-2-yl)-2,2-dimethyl-propionamide (0.5 g,5%). ¹H NMR (400 MHz, CDCl₃) δ 8.09 (d, J=8.0 Hz, 1H), 7.94 (br s, 1H),7.55 (d, J=8.4 Hz, 1H), 2.33 (s, 1H), 1.30 (s, 9H).

Step d: 6-Chloro-5-methyl-pyridin-2-ylamine

To N-(6-chloro-5-methyl-pyridin-2-yl)-2,2-dimethyl-propionamide (4.00 g,17.7 mmol) was added 6 N HCl (20 mL) at room temperature. The mixturewas stirred at 80° C. for 12 hours. The reaction mixture was basifiedwith drop-wise addition of sat. NaHCO₃ to pH 8-9, and then the mixturewas extracted with CH₂Cl₂ (3×). The organic phases were dried overNa₂SO₄ and evaporated under vacuum to obtain the6-chloro-5-methyl-pyridin-2-ylamine (900 mg, 36%). ¹H NMR (400 MHz,CDCl₃) δ 7.28 (d, J=8.0 Hz, 1H), 6.35 (d, J=8.0 Hz, 1H), 4.39 (br s,2H), 2.22 (s, 3H). MS (ESI) m/z: 143 (M+H⁺).

V. 6-Chloro-5-(trifluoromethyl)pyridin-2-amine

2,6-Dichloro-3-(trifluoromethyl)pyridine (5.00 g, 23.2 mmol) and 28%aqueous ammonia (150 mL) were placed in a 250 mL autoclave. The mixturewas heated at 93° C. for 21 h. The reaction was cooled to rt andextracted with EtOAc (100 mL×3). The combined organic extracts weredried over anhydrous Na₂SO₄ and evaporated under vacuum to give thecrude product, which was purified by column chromatography on silica gel(2-20% EtOAc in petroleum ether as eluant) to give6-chloro-5-(trifluoromethyl)pyridin-2-amine (2.1 g, 46% yield). ¹H NMR(400 MHz, DMSO-d₆) δ 7.69 (d, J=8.4 Hz, 1H), 7.13 (br s, 2H), 6.43 (d,J=8.4 Hz, 1H). MS (ESI) m/z (M+H)⁺197.2

General Procedure IV: Coupling Reactions

One equivalent of the appropriate carboxylic acid was placed in anoven-dried flask under nitrogen. Thionyl chloride (3 equivalents) and acatalytic amount of N,N-dimethylformamide was added and the solution wasallowed to stir at 60° C. for 30 minutes. The excess thionyl chloridewas removed under vacuum and the resulting solid was suspended in aminimum of anhydrous pyridine. This solution was slowly added to astirred solution of one equivalent the appropriate aminoheterocycledissolved in a minimum of anhydrous pyridine. The resulting mixture wasallowed to stir for 15 hours at 110° C. The mixture was evaporated todryness, suspended in dichloromethane, and then extracted three timeswith 1N NaOH. The organic layer was then dried over sodium sulfate,evaporated to dryness, and then purified by column chromatography.

W.1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-bromopyridin-2-yl)cyclopropane-carboxamide(B-1)

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (2.38 g, 11.5 mmol)was placed in an oven-dried flask under nitrogen. Thionyl chloride (2.5mL) and N,N-dimethylformamide (0.3 mL) were added and the solution wasallowed to stir for 30 minutes at 60° C. The excess thionyl chloride wasremoved under vacuum and the resulting solid was suspended in 7 mL ofanhydrous pyridine. This solution was then slowly added to a solution of5-bromo-pyridin-2-ylamine (2.00 g, 11.6 mmol) suspended in 10 mL ofanhydrous pyridine. The resulting mixture was allowed to stir for 15hours at 110° C. The mixture was then evaporated to dryness, suspendedin 100 mL of dichloromethane, and washed with three 25 mL portions of 1NNaOH. The organic layer was dried over sodium sulfate, evaporated tonear dryness, and then purified by silica gel column chromatographyutilizing dichloromethane as the eluent to yield the pure product (3.46g, 83%) ESI-MS m/z calc. 361.2. found 362.1 (M+1)⁺; Retention time 3.40minutes. ¹H NMR (400 MHz, DMSO-d₆) δ 1.06-1.21 (m, 2H), 1.44-1.51 (m,2H), 6.07 (s, 2H), 6.93-7.02 (m, 2H), 7.10 (d, J=1.6 Hz, 1H), 8.02 (d,J=1.6 Hz, 2H), 8.34 (s, 1H), 8.45 (s, 1H).

X.1-(Benzo[d][1,3]-dioxol-6-yl)-N-(6-bromopyridin-2-yl)cyclopropane-carboxamide(B-2)

(1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (1.2 g, 5.8 mmol)was placed in an oven-dried flask under nitrogen. Thionyl chloride (2.5mL) and N,N-dimethylformamide (0.3 mL) were added and the solution wasallowed to stir at 60° C. for 30 minutes. The excess thionyl chloridewas removed under vacuum and the resulting solid was suspended in 5 mLof anhydrous pyridine. This solution was then slowly added to a solutionof 6-bromopyridin-2-amine (1.0 g, 5.8 mmol) suspended in 10 mL ofanhydrous pyridine. The resulting mixture was allowed to stir for 15hours at 110° C. The mixture was then evaporated to dryness, suspendedin 50 mL of dichloromethane, and washed with three 20 mL portions of 1NNaOH. The organic layer was dried over sodium sulfate, evaporated tonear dryness, and then purified by silica gel column chromatographyutilizing dichloromethane containing 2.5% triethylamine as the eluent toyield the pure product. ESI-MS m/z calc. 361.2. found 362.1 (M+1)⁺;Retention time 3.43 minutes. ¹H NMR (400 MHz, DMSO-d₆) δ 1.10-1.17 (m,2H), 1.42-1.55 (m, 2H), 6.06 (s, 2H), 6.92-7.02 (m, 2H), 7.09 (d, J=1.6Hz, 1H), 7.33 (d, J=7.6 Hz, 1H), 7.73 (t, J=8.0 Hz, 1H), 8.04 (d, J=8.2Hz, 1H), 8.78 (s, 1H).

The compounds in the following Table 3 were prepared in a manneranalogous to that described above:

TABLE 3 Exemplary compounds synthesized according to Preparations W andX. ¹H NMR Retention (400 MHz, Compound Name Time (min) (M + 1)⁺ DMSO-d₆)B-3 1-(Benzo[d][1,3]dioxol-5- 3.58 375.3 ¹H NMR (400 MHz,yl)-N-(5-bromo-6- DMSO-d₆) δ methylpyridin-2- 8.39 (s, 1H),yl)cyclopropanecarboxamide 7.95 (d, J = 8.7 Hz, 1H), 7.83 (d, J = 8.8Hz, 1H), 7.10 (d, J = 1.6 Hz, 1H), 7.01-6.94 (m, 2H), 6.06 (s, 2H), 2.41(s, 3H), 1.48-1.46 (m, 2H), 1.14-1.10 (m, 2H) B-41-(Benzo[d][1,3]dioxol-5- 2.90 331.0 ¹H NMR (400 MHz, yl)-N-(6-chloro-5-DMSO-d₆) δ methylpyridin-2- 8.64 (s, 1H), yl)cyclopropanecarboxamide7.94-7.91 (m, 1H), 7.79-7.77 (m, 1H), 7.09 (m, 1H), 7.00-6.88 (m, 2H),6.06 (s, 2H), 2.25 (s, 3H), 1.47-1.44 (m, 2H), 1.13-1.10 (m, 2H) B-51-(Benzo[d][1,3]dioxol-5- 3.85 375.1 ¹H NMR (400 MHz, yl)-N-(5-bromo-4-DMSO-d₆) δ methylpyridin-2- 8.36 (s, 1H), yl)cyclopropanecarboxamide8.30 (s, 1H), 8.05 (s, 1H), 7.09 (d, J = 1.6 Hz, 1H), 7.01-6.95 (m, 2H),6.07 (s, 2H), 2.35 (s, 3H), 1.49-1.45 (m, 2H), 1.16-1.13 (m, 2H) B-61-(Benzo[d][1,3]dioxol-5- 3.25 389.3 ¹H NMR (400 MHz,yl)-N-(5-bromo-3,4- DMSO-d₆) δ dimethylpyridin-2- 8.82 (s, 1H),yl)cyclopropanecarboxamide 8.35 (s, 1H), 7.01 (m, 1H), 6.96-6.89 (m,2H), 6.02 (s, 2H), 2.35 (s, 3H), 2.05 (s, 3H), 1.40-1.38 (m, 2H),1.08-1.05 (m, 2H) B-7 1-(Benzo[d][1,3]dioxol-5- 2.91 375.1yl)-N-(5-bromo-3- methylpyridin-2- yl)cyclopropanecarboxamide B-81-(Benzo[d][1,3]dioxol-5- 2.88 318.3 ¹H NMR (400 MHz,yl)-N-(6-chloropyridazin-3- DMSO-d₆) δ yl)cyclopropanecarboxamide1.15-1.19 (m, 2H), 1.48-1.52 (m, 2H), 6.05 (s, 2H), 6.93-7.01 (m, 2H),7.09 (d, J = 1.7 Hz, 1H), 7.88 (d, J = 9.4 Hz, 1H), 8.31 (d, J = 9.4 Hz,1H), 9.46 (s, 1H) B-9 1-(Benzo[d][1,3]dioxol-5- 3.20 318.3 ¹H NMR (400MHz, yl)-N-(5-bromopyrazin-2- DMSO-d₆) δ yl)cyclopropanecarboxamide1.13-1.18 (m, 2H), 1.47-1.51 (m, 2H), 6.04 (s, 2H), 6.90-6.99 (m, 2H),7.06 (d, J = 1.6 Hz, 1H),, 8.47 (s, 1H), 9.21 (s, 1H), 9.45 (s, 1H) B-101-(Benzo[d][1,3]dioxol-5- 3.45 362.1 ¹H NMR (400 MHz,yl)-N-(6-chloropyrazin-2- DMSO-d₆) δ yl)cyclopropanecarboxamide1.12-1.23 (m, 2H), 1.41-1.58 (m, 2H), 6.04 (s, 2H), 6.90-7.00 (m, 2H),7.07 (d, J = 1.6 Hz, 1H), 8.55 (s, 1H), 8.99-9.21 (m, 2H) B-11N-(6-bromopyridin-2-yl)-1- 2.12 397.3 ¹H NMR (400 MHz, (2,2- DMSO-d₆) δdifluorobenzo[d][1,3]dioxol- 9.46 (s, 1H), 5- 8.01-7.99 (m, 1H),yl)cyclopropanecarboxamide 7.75-7.71 (m, 1H), 7.54 (m, 1H), 7.41-7.39(m, 1H), 7.36-7.30 (m, 2H), 1.52-1.49 (m, 2H), 1.20-1.17 (m, 2H) B-12N-(6-chloro-5- 2.18 367.1 ¹H NMR (400 MHz, methylpyridin-2-yl)-1-(2,2-DMSO-d₆) δ difluorobenzo[d][1,3]dioxol- 9.30 (s, 1H), 5- 7.89-7.87 (m,1H), yl)cyclopropanecarboxamide 7.78-7.76 (m, 1H), 7.53 (m, 1H),7.41-7.39 (m, 1H), 7.33-7.30 (m, 1H), 2.26 (s, 3H), 1.51-1.49 (m, 2H),1.18-1.16 (m, 2H) B-13 N-(6-chloro-5- 1.98 421.1 ¹H NMR (400 MHz,(trifluoromethyl)pyridin-2- DMSO-d₆) δ yl)-1-(2,2- 10.09 (s, 1H),difluorobenzo[d][1,3]dioxol- 8.29 (m, 1H), 8.16 (m, 5- 1H), 7.53 (m,1H), yl)cyclopropanecarboxamide 7.41-7.38 (m, 1H), 7.34-7.29 (m, 1H),1.56-1.53 (m, 2H), 1.24-1.22 (m, 2H)

General Procedure V: Compounds of Formula I

The appropriate aryl halide (1 equivalent) was dissolved in 1 mL ofN,N-dimethylformamide (DMF) in a reaction tube. The appropriate boronicacid (1.3 equivalents), 0.1 mL of an aqueous 2 M potassium carbonatesolution (2 equivalents), and a catalytic amount of Pd(dppf)Cl₂ (0.09equivalents) were added and the reaction mixture was heated at 80° C.for three hours or at 150° C. for 5 min in the microwave. The resultingmaterial was cooled to room temperature, filtered, and purified byreverse-phase preparative liquid chromatography.

Y. 1-Benzo[1,3]-dioxol-5-yl-cyclopropanecarboxylic acid[5-(2,4-dimethoxy-phenyl)-pyridin-2-yl]-amide

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid(5-bromo-pyridin-2-yl)-amide (36.1 mg, 0.10 mmol) was dissolved in 1 mLof N,N-dimethylformamide in a reaction tube. 2,4-Dimethoxybenzeneboronicacid (24 mg, 0.13 mmol), 0.1 mL of an aqueous 2 M potassium carbonatesolution, and a catalytic amount of Pd(dppf)Cl₂ (6.6 mg, 0.0090 mmol)were added and the reaction mixture was heated at 80° C. for threehours. The resulting material was cooled to room temperature, filtered,and purified by reverse-phase preparative liquid chromatography to yieldthe pure product as a trifluoroacetic acid salt. ESI-MS m/z calc. 418.2.found 419.0 (M+1)⁺. Retention time 3.18 minutes. ¹H NMR (400 MHz, CD₃CN)δ 1.25-1.29 (m, 2H), 1.63-1.67 (m, 2H), 3.83 (s, 3H), 3.86 (s, 3H), 6.04(s, 2H), 6.64-6.68 (m, 2H), 6.92 (d, J=8.4 Hz, 1H), 7.03-7.06 (m, 2H),7.30 (d, J=8.3 Hz, 1H), 7.96 (d, J=8.9 Hz, 1H), 8.14 (dd, J=8.9, 2.3 Hz,1H), 8.38 (d, J=2.2 Hz, 1H), 8.65 (s, 1H).

Z. 1-Benzo[1,3]-dioxol-5-yl-cyclopropanecarboxylicacid[6-(4-dimethylamino-phenyl)-pyridin-2-yl]-amide

1-Benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid(6-bromo-pyridin-2-yl)-amide (36 mg, 0.10 mmol) was dissolved in 1 mL ofN,N-dimethylformamide in a reaction tube. 4-(Dimethylamino)phenylboronicacid (21 mg, 0.13 mmol), 0.1 mL of an aqueous 2 M potassium carbonatesolution, and (Pd(dppf)Cl₂ (6.6 mg, 0.0090 mmol) were added and thereaction mixture was heated at 80° C. for three hours. The resultingmaterial was cooled to room temperature, filtered, and purified byreverse-phase preparative liquid chromatography to yield the pureproduct as a trifluoroacetic acid salt. ESI-MS m/z calc. 401.2. found402.5 (M+1)⁺. Retention time 2.96 minutes. ¹H NMR (400 MHz, CD₃CN) δ1.23-1.27 (m, 2H), 1.62-1.66 (m, 2H), 3.04 (s, 6H), 6.06 (s, 2H),6.88-6.90 (m, 2H), 6.93-6.96 (m, 1H), 7.05-7.07 (m, 2H), 7.53-7.56 (m,1H), 7.77-7.81 (m, 3H), 7.84-7.89 (m, 1H), 8.34 (s, 1H).

The following schemes were utilized to prepare additional boronic esterswhich were not commercially available:

AA.1-Methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-sulfonylpiperazine

Step a: 1-(4-Bromophenylsulfonyl)-4-methylpiperazine

A solution of 4-bromobenzene-1-sulfonyl chloride (256 mg, 1.00 mmol) in1 mL of dichloromethane was slowly added to a vial (40 mL) containing 5mL of a saturated aqueous solution of sodium bicarbonate,dichloromethane (5 mL) and 1-methylpiperazine (100 mg, 1.00 mmol). Thereaction was stirred at room temperature overnight. The phases wereseparated and the organic layer was dried over magnesium sulfate.Evaporation of the solvent under reduced pressure provided the requiredproduct, which was used in the next step without further purification.ESI-MS m/z calc. 318.0. found 318.9 (M+1)⁺. Retention time of 1.30minutes. ¹H NMR (300 MHz, CDCl₃) δ 7.65 (d, J=8.7 Hz, 2H), 7.58 (d,J=8.7 Hz, 2H), 3.03 (t, J=4.2 Hz, 4H), 2.48 (t, J=4.2 Hz, 4H), 2.26 (s,3H).

Step b:1-Methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonyl-piperazine

A 50 mL round bottom flask was charged with1-(4-bromophenyl-sulfonyl)-4-methylpiperazine (110 mg, 0.350 mmol),bis-(pinacolato)-diboron (93 mg, 0.37 mmol), palladium acetate (6 mg,0.02 mmol), and potassium acetate (103 mg, 1.05 mmol) inN,N-dimethylformamide (6 mL). The mixture was degassed by gentlybubbling argon through the solution for 30 minutes at room temperature.The mixture was then heated at 80° C. under argon until the reaction wascomplete (4 hours). The desired product,1-methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-sulfonyl-piperazine,and the bi-aryl product,4-(4-methylpiperazin-1-ylsulfonyl)-phenyl-phenylsulfonyl-4-methylpiperazine,were obtained in a ratio of 1:2 as indicated by LC/MS analysis. Themixture was used without further purification.

BB.4,4,5,5-Tetramethyl-2-(4-(2-(methylsulfonyl)ethyl)phenyl)-1,3,2-dioxaborolane

Step a: 4-Bromophenethyl-4-methylbenzenesulfonate

To a 50 mL round-bottom flask was added p-bromophenethyl alcohol (1.0 g,4.9 mmol), followed by the addition of pyridine (15 mL). To this clearsolution was added, under argon, p-toluenesulfonyl chloride (TsCl) (1.4g, 7.5 mmol) as a solid. The reaction mixture was purged with Argon andstirred at room temperature for 18 hours. The crude mixture was treatedwith 1N HCl (20 mL) and extracted with ethyl acetate (5×25 mL). Theorganic fractions were dried over Na₂SO₄, filtered, and concentrated toyield 4-bromophenethyl-4-methylbenzenesulfonate (0.60 g, 35%) as ayellowish liquid. ¹H-NMR (Acetone-d₆, 300 MHz) δ 7.64 (d, J=8.4 Hz, 2H),7.40-7.37 (d, J=8.7 Hz, 4H), 7.09 (d, J=8.5 Hz, 2H), 4.25 (t, J=6.9 Hz,2H), 2.92 (t, J=6.3 Hz, 2H), 2.45 (s, 3H).

Step b: (4-Bromophenethyl)(methyl)sulfane

To a 20 mL round-bottom flask were added 4-bromophenethyl4-methylbenzenesulfonate (0.354 g, 0.996 mmol) and CH₃SNa (0.10 g, 1.5mmol), followed by the addition of THF (1.5 mL) andN-methyl-2-pyrrolidinone (1.0 mL). The mixture was stirred at roomtemperature for 48 hours, and then treated with a saturated aqueoussolution of sodium bicarbonate (10 mL). The mixture was extracted withethyl acetate (4×10 mL), dried over Na₂SO₄, filtered, and concentratedto yield (4-bromophenethyl)(methyl)sulfane (0.30 g crude) as a yellowishoil. ¹H-NMR (CDCl₃, 300 MHz) δ 7.40 (d, J=8.4 Hz, 2H), 7.06 (d, J=8.4Hz, 2H), 2.89-2.81 (m, 2H), 2.74-2.69 (m, 2H), 2.10 (s, 3H).

Step c: 1-Bromo-4-(2-methylsulfonyl)-ethylbenzene

To a 20 mL round-bottom flask were added(4-bromophenethyl)-(methyl)sulfane (0.311 g, 1.34 mmol) and Oxone (3.1g, 0.020 mol), followed by the addition of a 1:1 mixture ofacetone/water (10 mL). The mixture was vigorously stirred at roomtemperature for 20 hours, before being concentrated. The aqueous mixturewas extracted with ethyl acetate (3×15 mL) and dichloromethane (3×10mL). The organic fractions were combined, dried with Na₂SO₄, filtered,and concentrated to yield a white semisolid. Purification of the crudematerial by flash chromatography yielded1-bromo-4-(2-methylsulfonyl)-ethylbenzene (0.283 g, 80%). ¹H-NMR(DMSO-d₆, 300 MHz) δ 7.49 (d, J=8.4 Hz, 2H), 7.25 (d, J=8.7 Hz, 2H),3.43 (m, 2H), 2.99 (m, 2H), 2.97 (s, 3H).

Step d:4,4,5,5-Tetramethyl-2-(4-(2-(methylsulfonyl)ethyl)-phenyl)-1,3,2-dioxaborolane

4,4,5,5-Tetramethyl-2-(4-(2-(methylsulfonyl)ethyl)phenyl)-1,3,2-dioxaborolanewas prepared in the same manner as described above for1-methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonyl-piperazine,Preparation AA.

CC. tert-Butylmethyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)carbamate

Step a: tert-Butyl-4-bromobenzylcarbamate

Commercially available p-bromobenzylamine hydrochloride (1 g, 4 mmol)was treated with 10% aq. NaOH (5 mL). To the clear solution was added(Boc)₂O (1.1 g, 4.9 mmol) dissolved in dioxane (10 mL). The mixture wasvigorously stirred at room temperature for 18 hours. The resultingresidue was concentrated, suspended in water (20 mL), extracted withethyl acetate (4×20 mL), dried over Na₂SO₄, filtered, and concentratedto yield tert-butyl-4-bromobenzylcarbamate (1.23 g, 96%) as a whitesolid. ¹H NMR (300 MHz, DMSO-d₆) δ 7.48 (d, J=8.4 Hz, 2H), 7.40 (t, J=6Hz, 1H), 7.17 (d, J=8.4 Hz, 2H), 4.07 (d, J=6.3 Hz, 2H), 1.38 (s, 9H).

Step b: tert-Butyl-4-bromobenzyl(methyl)carbamate

In a 60-mL vial, tert-butyl-4-bromobenzylcarbamate (1.25 g, 4.37 mmol)was dissolved in DMF (12 mL). To this solution was added Ag₂O (4.0 g, 17mmol) followed by the addition of CH₃I (0.68 mL, 11 mmol). The mixturewas stirred at 50° C. for 18 hours. The reaction mixture was filteredthrough a bed of celite and the celite was washed with methanol (2×20mL) and dichloromethane (2×20 mL). The filtrate was concentrated toremove most of the DMF. The residue was treated with water (50 mL) and awhite emulsion formed. This mixture was extracted with ethyl acetate(4×25 mL), dried over Na₂SO₄, and the solvent was evaporated to yieldtert-butyl-4-bromobenzyl(methyl)carbamate (1.3 g, 98%) as a yellow oil.¹H NMR (300 MHz, DMSO-d₆) δ 7.53 (d, J=8.1 Hz, 2H), 7.15 (d, J=8.4 Hz,2H), 4.32 (s, 2H), 2.74 (s, 3H), 1.38 (s, 9H).

Step c: tert-Butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylmethylcarbamate

The coupling reaction was achieved in the same manner as described abovefor1-methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonyl-piperazine,Preparation AA. The Boc protecting group was removed after the couplingreaction by treating the crude reaction mixture with 0.5 mL of 1N HCl indiethyl ether for 18 hours before purification by HPLC.

Additional examples of the invention were prepared following the aboveprocedure with non-substantial changes but using aryl boronic acidsgiven in Table 4.

TABLE 4 Additional exemplary compounds of formula I. Compound No. AmineBoronic Acid 1 B-2 [2-(dimethylaminomethyl)phenyl]boronic acid 2 B-2[4-(1-piperidyl)phenyl]boronic acid 3 B-2 (3,4-dichlorophenyl)boronicacid 4 B-2 (4-morpholinosulfonylphenyl)boronic acid 5 B-2(3-chloro-4-methoxy-phenyl)boronic acid 6 B-2(6-methoxy-3-pyridyl)boronic acid 7 B-2 (4-dimethylaminophenyl)boronicacid 8 B-2 (4-morpholinophenyl)boronic acid 9 B-2[4-(acetylaminomethyl)phenyl]boronic acid 10 B-2(2-hydroxyphenyl)boronic acid 11 B-1 2-dihydroxyboranylbenzoic acid 12B-1 (6-methoxy-3-pyridyl)boronic acid 14 B-2 (2,4-dimethylphenyl)boronicacid 15 B-2 [3-(hydroxymethyl)phenyl]boronic acid 16 B-23-dihydroxyboranylbenzoic acid 17 B-2 (3-ethoxyphenyl)boronic acid 18B-2 (3,4-dimethylphenyl)boronic acid 19 B-1[4-(hydroxymethyl)phenyl]boronic acid 20 B-1 3-pyridylboronic acid 21B-2 (4-ethylphenyl)boronic acid 23 B-2 4,4,5,5-tetramethyl-2-(4-(2-(methylsulfonyl)ethyl)phenyl)-1,3,2- dioxaborolane 24 B-1benzo[1,3]dioxol-5-ylboronic acid 25 B-2 (3-chlorophenyl)boronic acid 26B-2 (3-methylsulfonylaminophenyl)boronic acid 27 B-2(3,5-dichlorophenyl)boronic acid 28 B-2 (3-methoxyphenyl)boronic acid 29B-1 (3-hydroxyphenyl)boronic acid 31 B-2 phenylboronic acid 32 B-2(2,5-difluorophenyl)boronic acid 33 B-8 phenylboronic acid 36 B-2(2-methylsulfonylaminophenyl)boronic acid 37 B-1 1H-indol-5-ylboronicacid 38 B-2 2,2,2-trifluoro-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)acetamide 39 B-2 (2-chlorophenyl)boronic acid40 B-1 m-tolylboronic acid 41 B-2 (2,4-dimethoxypyrimidin-5-yl)boronicacid 42 B-2 (4-methoxycarbonylphenyl)boronic acid 43 B-2 tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylmethylcarbamate^((a)) 44 B-2(4-ethoxyphenyl)boronic acid 45 B-2 (3-methylsulfonylphenyl)boronic acid46 B-2 (4-fluoro-3-methyl-phenyl)boronic acid 47 B-2(4-cyanophenyl)boronic acid 48 B-1 (2,5-dimethoxyphenyl)boronic acid 49B-1 (4-methylsulfonylphenyl)boronic acid 50 B-1 cyclopent-1-enylboronicacid 51 B-2 o-tolylboronic acid 52 B-1 (2,6-dimethylphenyl)boronic acid53 B-8 2-chlorophenylboronic acid 54 B-2 (2,5-dimethoxyphenyl)boronicacid 55 B-2 (2-fluoro-3-methoxy-phenyl)boronic acid 56 B-2(2-methoxyphenyl)boronic acid 57 B-9 phenylboronic acid 58 B-2(4-isopropoxyphenyl)boronic acid 59 B-2 (4-carbamoylphenyl)boronic acid60 B-2 (3,5-dimethylphenyl)boronic acid 61 B-2 (4-isobutylphenyl)boronicacid 62 B-1 (4-cyanophenyl)boronic acid 63 B-10 phenylboronic acid 64B-2 N-ethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-benzenesulfonamide 65 B-12,3-dihydrobenzofuran-5-ylboronic acid 66 B-2 (4-chlorophenyl)boronicacid 67 B-2 (4-chloro-3-methyl-phenyl)boronic acid 68 B-2(2-fluorophenyl)boronic acid 69 B-2 benzo[1,3]dioxol-5-ylboronic acid 70B-2 (4-morpholinocarbonylphenyl)boronic acid 71 B-1cyclohex-1-enylboronic acid 72 B-2 (3,4,5-trimethoxyphenyl)boronic acid73 B-2 [4-(dimethylaminomethyl)phenyl]boronic acid 74 B-2 m-tolylboronicacid 77 B-2 (3-cyanophenyl)boronic acid 78 B-2[3-(tert-butoxycarbonylaminomethyl)- phenyl]boronicacid^((a)) 79 B-2(4-methylsulfonylphenyl)boronic acid 80 B-1 p-tolylboronic acid 81 B-2(2,4-dimethoxyphenyl)boronic acid 82 B-2(2-methoxycarbonylphenyl)boronic acid 83 B-2 (2,4-difluorophenyl)boronicacid 84 B-2 (4-isopropylphenyl)boronic acid 85 B-2[4-(2-dimethylaminoethylcarbamoyl)- phenyl]boronic acid 86 B-1(2,4-dimethoxyphenyl)boronic acid 87 B-1 benzofuran-2-ylboronic acid 88B-2 2,3-dihydrobenzofuran-5-ylboronic acid 89 B-2(3-fluoro-4-methoxy-phenyl)boronic acid 91 B-1 (3-cyanophenyl)boronicacid 92 B-1 (4-dimethylaminophenyl)boronic acid 93 B-2(2,6-dimethoxyphenyl)boronic acid 94 B-2(2-methoxy-5-methyl-phenyl)boronic acid 95 B-2(3-acetylaminophenyl)boronic acid 96 B-1(2,4-dimethoxypyrimidin-5-yl)boronic acid 97 B-2(5-fluoro-2-methoxy-phenyl)boronic acid 98 B-1[3-(hydroxymethyl)phenyl]boronic acid 99 B-1 (2-methoxyphenyl)boronicacid 100 B-2 (2,4,6-trimethylphenyl)boronic acid 101 B-2[4-(dimethylcarbamoyl)phenyl]boronic acid 102 B-2[4-(tert-butoxycarbonylaminomethyl)- phenyl]boronic acid^((a)) 104 B-1(2-chlorophenyl)boronic acid 105 B-1 (3-acetylaminophenyl)boronic acid106 B-2 (2-ethoxyphenyl)boronic acid 107 B-2 3-furylboronic acid 108 B-2[2-(hydroxymethyl)phenyl]boronic acid 110 B-9 2-chlorophenylboronic acid111 B-2 (2-fluoro-6-methoxy-phenyl)boronic acid 112 B-2(2-ethoxy-5-methyl-phenyl)boronic acid 113 B-2 1H-indol-5-ylboronic acid114 B-1 (3-chloro-4-pyridyl)boronic acid 115 B-2 cyclohex-1-enylboronicacid 116 B-1 o-tolylboronic acid 119 B-2 (2-aminophenyl)boronic acid 120B-2 (4-methoxy-3,5-dimethyl-phenyl)boronic acid 121 B-2(4-methoxyphenyl)boronic acid 122 B-2 (2-propoxyphenyl)boronic acid 123B-2 (2-isopropoxyphenyl)boronic acid 124 B-2 (2,3-dichlorophenyl)boronicacid 126 B-2 (2,3-dimethylphenyl)boronic acid 127 B-2(4-fluorophenyl)boronic acid 128 B-1 (3-methoxyphenyl)boronic acid 129B-2 (4-chloro-2-methyl-phenyl)boronic acid 130 B-1(2,6-dimethoxyphenyl)boronic acid 131 B-2(5-isopropyl-2-methoxy-phenyl)boronic acid 132 B-2(3-isopropoxyphenyl)boronic acid 134 B-2 4-dihydroxyboranylbenzoic acid135 B-2 (4-dimethylamino-2-methoxy-phenyl)boronic acid 136 B-2(4-methylsulfinylphenyl)boronic acid 137 B-2[4-(methylcarbamoyl)phenyl]boronic acid 138 B-1 8-quinolylboronic acid139 B-2 cyclopent-1-enylboronic acid 140 B-2 p-tolylboronic acid 142 B-82-methoxyphenylboronic acid 143 B-2 (2,5-dimethylphenyl)boronic acid 144B-1 (3,4-dimethoxyphenyl)boronic acid 145 B-1 (3-chlorophenyl)boronicacid 146 B-2 [4-(morpholinomethyl)phenyl]boronic acid 147 B-104-(dimethylamino)phenylboronic acid 148 B-2[4-(methylsulfamoyl)phenyl]boronic acid 149 B-14-dihydroxyboranylbenzoic acid 150 B-1 phenylboronic acid 151 B-2(2,3-difluorophenyl)boronic acid 152 B-1 (4-chlorophenyl)boronic acid153 B-9 2-methoxyphenylboronic acid 154 B-2 3-dihydroxyboranylbenzoicacid 155 B-10 2-methoxyphenylboronic acid 157 B-2(3-chloro-4-fluoro-phenyl)boronic acid 158 B-2(2,3-dimethoxyphenyl)boronic acid 159 B-2[4-(tert-butoxycarbonylaminomethyl)- phenyl]boronic acid 160 B-2(4-sulfamoylphenyl)boronic acid 161 B-2 (3,4-dimethoxyphenyl)boronicacid 162 B-2 [4-(methylsulfonylamino- methyl)phenyl]boronic acid 166 B-14-(N,N-dimethylsulfamoyl)phenylboronic acid 167 B-62-isopropylphenylboronic acid 171 B-6 4-(methylcarbamoyl)phenylboronicacid 173 B-2 3-fluorophenylboronic acid 174 B-63-(N,N-dimethylsulfamoyl)phenylboronic acid 179 B-64-(N-methylsulfamoyl)phenylboronic acid 181 B-13-((tert-butoxycarbonylamino)methyl)- phenylboronic acid 185 B-33-methoxyphenylboronic acid 186 B-6 2-chlorophenylboronic acid 187 B-73-(dimethylcarbamoyl)phenylboronic acid 188 B-63-(hydroxymethyl)phenylboronic acid 189 B-13-(N,N-dimethylsulfamoyl)phenylboronic acid 190 B-14-sulfamoylphenylboronic acid 191 B-1 2-isopropylphenylboronic acid 193B-5 3-sulfamoylphenylboronic acid 194 B-3 4-isopropylphenylboronic acid195 B-3 3-(N,N-dimethylsulfamoyl)phenylboronic acid 196 B-74-(methylcarbamoyl)phenylboronic acid 198 B-33-(dimethylcarbamoyl)phenylboronic acid 204 B-53-(dimethylcarbamoyl)phenylboronic acid 206 B-3 4-chlorophenylboronicacid 207 B-1 4-(N-methylsulfamoyl)phenylboronic acid 209 B-13-(methylcarbamoyl)phenylboronic acid 210 B-3 4-sulfamoylphenylboronicacid 213 B-5 3-isopropylphenylboronic acid 215 B-74-methoxyphenylboronic acid 216 B-6 3-chlorophenylboronic acid 217 B-7m-tolylboronic acid 219 B-5 4-(hydroxymethyl)phenylboronic acid 222 B-6m-tolylboronic acid 224 B-5 2-chlorophenylboronic acid 225 B-13-isopropylphenylboronic acid 227 B-6 4-(hydroxymethyl)phenylboronicacid 229 B-7 3-chlorophenylboronic acid 230 B-6 o-tolylboronic acid 231B-1 2-(hydroxymethyl)phenylboronic acid 235 B-3 3-isopropylphenylboronicacid 238 B-5 3-carbamoylphenylboronic acid 241 B-24-(N,N-dimethylsulfamoyl)phenylboronic acid 243 B-72-methoxyphenylboronic acid 247 B-6 3-(dimethylcarbamoyl)phenylboronicacid 251 B-3 3-sulfamoylphenylboronic acid 252 B-14-methoxyphenylboronic acid 254 B-3 4-(N-methylsulfamoyl)phenylboronicacid 255 B-1 4-((tert-butoxycarbonylamino)methyl)- phenylboronic acid257 B-5 4-chlorophenylboronic acid 258 B-33-(methylcarbamoyl)phenylboronic acid 260 B-32-(hydroxymethyl)phenylboronic acid 263 B-44-(hydroxymethyl)phenylboronic acid 264 B-7 4-chlorophenylboronic acid265 B-6 4-carbamoylphenylboronic acid 266 B-5 3-methoxyphenylboronicacid 269 B-7 phenylboronic acid 272 B-3 4-methoxyphenylboronic acid 274B-6 2-(hydroxymethyl)phenylboronic acid 277 B-34-(hydroxymethyl)phenylboronic acid 278 B-33-(methylcarbamoyl)phenylboronic acid 280 B-34-(N,N-dimethylsulfamoyl)phenylboronic acid 283 B-34-carbamoylphenylboronic acid 286 B-1 4-(methylcarbamoyl)phenylboronicacid 287 B-2 4-(trifluoromethoxy)phenylboronic acid 288 B-54-(N-methylsulfamoyl)phenylboronic acid 289 B-3 phenylboronic acid 290B-6 4-isopropylphenylboronic acid 291 B-3 3-(hydroxymethyl)phenylboronicacid 293 B-6 3-methoxyphenylboronic acid 294 B-72-(hydroxymethyl)phenylboronic acid 295 B-3 3-carbamoylphenylboronicacid 296 B-5 m-tolylboronic acid 297 B-14-(dimethylcarbamoyl)phenylboronic acid 298 B-3 2-methoxyphenylboronicacid 299 B-7 p-tolylboronic acid 300 B-3 o-tolylboronic acid 301 B-52-(hydroxymethyl)phenylboronic acid 303 B-6 2-methoxyphenylboronic acid305 B-6 3-isopropylphenylboronic acid 308 B-7 4-isopropylphenylboronicacid 309 B-3 4-(dimethylcarbamoyl)phenylboronic acid 310 B-54-(methylcarbamoyl)phenylboronic acid 313 B-7 o-tolylboronic acid 314B-7 3-(methylcarbamoyl)phenylboronic acid 315 B-3 p-tolylboronic acid320 B-1 3-(dimethylcarbamoyl)phenylboronic acid 321 B-54-sulfamoylphenylboronic acid 322 B-6 phenylboronic acid 323 B-5o-tolylboronic acid 324 B-3 4-((tert-butoxycarbonylamino)methyl)-phenylboronic acid^((a)) 326 B-5 4-(dimethylcarbamoyl)phenylboronic acid327 B-5 2-methoxyphenylboronic acid 328 B-1 4-isopropylphenylboronicacid 329 B-5 2-isopropylphenylboronic acid 331 B-3 m-tolylboronic acid333 B-6 4-methoxyphenylboronic acid 334 B-5 4-methoxyphenylboronic acid337 B-6 p-tolylboronic acid 343 B-54-(N,N-dimethylsulfamoyl)phenylboronic acid 346 B-32-isopropylphenylboronic acid 348 B-64-((tert-butoxycarbonylamino)methyl)phenyl- boronic acid^((a)) 349 B-13-sulfamoylphenylboronic acid 350 B-33-((tert-butoxycarbonylamino)methyl)phenyl- boronic acid^((a)) 351 B-5phenylboronic acid 352 B-7 2-isopropylphenylboronic acid 353 B-64-chlorophenylboronic acid 354 B-7 2-chlorophenylboronic acid 355 B-53-(N,N-dimethylsulfamoyl)phenylboronic acid 356 B-73-sulfamoylphenylboronic acid 357 B-7 4-(N-methylsulfamoyl)phenylboronicacid 359 B-1 4-carbamoylphenylboronic acid 361 B-3 3-chlorophenylboronicacid 365 B-1 3-carbamoylphenylboronic acid 367 B-73-(hydroxymethyl)phenylboronic acid 368 B-44-(dimethylcarbamoyl)phenylboronic acid 370 B-53-(hydroxymethyl)phenylboronic acid 371 B-53-(methylcarbamoyl)phenylboronic acid 374 B-6 4-sulfamoylphenylboronicacid 375 B-5 4-carbamoylphenylboronic acid 389 B-122-methyl-3-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid390 B-11 3-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 391 B-134-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoic acid 392 B-113-methyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid393 B-12 2-chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 394 B-123-methoxy-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid395 B-2 4-cyclohexylphenylboronic acid 396 B-123-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoic acid 397 B-113-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoic acid 398 B-123-fluoro-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid399 B-13 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 400 B-133-fluoro-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid401 B-11 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 402 B-122-methoxy-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid403 B-11 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 404 B-112-methoxy-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid405 B-12 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 406 B-132-fluoro-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid407 B-11 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoic acid408 B-13 2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 410 B-24-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)aniline 411 B-133-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoic acid 412 B-22-methoxypyridin-3-ylboronic acid 414 B-113-fluoro-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid415 B-13 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid 417 B-122-fluoro-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid418 B-4 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoic acid 419B-11 2-chloro-5-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoicacid 420 B-2 4-(hydroxymethyl)phenylboronic acid 421 B-112-fluoro-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzoic acid422 B-12 3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid ^((a))The Boc protecting group wasremoved after the coupling reaction by treating the crude reactionmixture with 0.5 mL of 1N HCl in diethyl ether for 18 hours beforepurification by HPLC.

Further examples of the invention may be prepared by modification ofintermediates as illustrated above.

Compound Derivatization after Coupling:

DD. 1-(Benzo[d][1,3]-dioxol-5-yl)-N-(6 (4 (2methylpyrrolidin-1-ylsulfonyl)phenyl)pyridin-2-yl)cyclopropanecarboxamide

Step a: 4-(4,4′-Dimethoxybenzhydryl)-thiophenyl boronic acid

4,4′-Dimethoxybenzhydrol (2.7 g, 11 mmol) and 4-mercaptophenylboronicacid (1.54 g, 10 mmol) were dissolved in 20 mL AcOH and heated at 60° C.for 1 h. Solvent was evaporated and the residue was dried under highvacuum. This material was used without further purification.

Step b: 6-(4-(Bis(4-methoxyphenyl)methylthio)phenyl)pyridin-2-amine

4-(4,4′-Dimethoxybenzhydryl)-thiophenyl boronic acid (10 mmol) and2-amino-6-bromopyridine (1.73 g, 10 mmol) were dissolved in MeCN (40 mL)followed by addition of Pd(PPh₃)₄ (˜50 mg) and aq. K₂CO₃ (1M, 22 mL).The reaction mixture was heated portion wise in a microwave oven (160°C., 400 sec). The products were distributed between ethyl acetate andwater. The organic layer was washed with water, brine and dried overMgSO₄. Evaporation of the volatiles yielded an oil that was used withoutpurification in the next step. ESI-MS m/z calc. 428.0. found 429.1(M+1).

Step c:1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-(4-(bis(4-methoxyphenyl)methylthio)phenyl)-pyridin-2-yl)cyclopropanecarboxamide

6-[(4,4′-Dimethoxybenzhydryl)-4-thiophenyl]pyridin-2-ylamine (˜10 mmol)and 1-benzo[1,3]dioxol-5-yl-cyclopropanecarboxylic acid (2.28 g, 11mmol) were dissolved in chloroform (25 mL) followed by the addition ofTCPH (4.1 g, 12 mmol) and DIEA (5 mL, 30 mmol). The reaction mixture washeated at 65° C. for 48 h before the volatiles were removed underreduced pressure. The residue was transferred to a separatory funnel anddistributed between water (200 mL) and ethyl acetate (150 mL). Theorganic layer was washed with 5% NaHCO₃ (2×150 mL), water (1×150 mL),brine (1×150 mL) and dried over MgSO₄. Evaporation of the solventyielded crude1-(benzo[d][1,3]dioxol-5-yl)-N-(6-(4-(bis(4-methoxyphenyl)-methylthio)phenyl)pyridin-2-yl)cyclopropanecarboxamideas a pale oil. ESI-MS m/z calc. 616.0. found 617.0 (M+1) (HPLC purity˜85%, UV254 nm).

Step d:4-(6-(1-(Benzo[d][1,3]dioxol-5-yl)cyclopropane-carboxamido)pyridin-2-yl)benzenesulfonicacid

1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-(4-(bis(4-methoxyphenyl)methylthio)-phenyl)pyridin-2-yl)cyclopropanecarboxamide(˜8.5 mmol) was dissolved in AcOH (75 mL) followed by the addition of30% H₂O₂ (10 mL). Additional hydrogen peroxide (10 ml) was added 2 hlater. The reaction mixture was stirred at 35-45° C. overnight (˜90%conversion, HPLC). The volume of reaction mixture was reduced to a thirdby evaporation (bath temperature below 40° C.). The reaction mixture wasloaded directly onto a prep RP HPLC column (C-18) and purified.Fractions with4-(6-(1-(benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-2-yl)benzenesulfonicacid were collected and evaporated (1.9 g, 43%, cal. based on4-mercaptophenylboronic acid). ESI-MS m/z calc. 438.0. found 438.9(M+1).

Step e:4-(6-(1-(Benzo[d][1,3]dioxol-5-yl)cyclopropane-carboxamido)pyridin-2-yl)benzene-1-sulfonylchloride

4-(6-(1-(Benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-2-yl)benzenesulfonicacid (1.9 g, 4.3 mmol) was dissolved in POCl₃ (30 mL) followed by theaddition of SOCl₂ (3 mL) and DMF (100 μl). The reaction mixture washeated at 70-80° C. for 15 min. The volatiles were evaporated and thenre-evaporated with chloroform-toluene. The residual brown oil wasdiluted with chloroform (22 mL) and used for sulfonylation immediately.ESI-MS m/z calc. 456.0. found 457.1 (M+1).

Step f:1-(Benzo[d][1,3]dioxol-5-yl)-N-(6-(4-(2-methylpyrrolidin-1-ylsulfonyl)phenyl)pyridin-2-yl)cyclopropanecarboxamide

4-(6-(1-(Benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)pyridin-2-yl)benzene-1-sulfonylchloride (˜35 μmol, 400 μl solution in chloroform) was treated with2-methylpyrrolidine followed by the addition of DIEA (100 μl). Thereaction mixture was kept at room temperature for 1 h, concentrated,then diluted with DMSO (400 μl). The resulting solution was subjected toHPLC purification. Fractions containing the desired material werecombined and concentrated in vacuum centrifuge at 40° C. to provide thetrifluoroacetic salt of target material (ESI-MS m/z calc. 505.0. found505.9 (M+1), retention time 4.06 min) ¹H NMR (250 MHz, DMSO-d₆) δ 1.15(m. 2H), δ 1.22 (d, 3H, J=6.3 Hz), δ 1.41-1.47 (m, 2H), δ 1.51 (m, 2H),δ 1.52-1.59 (m, 2H), δ 3.12 (m, 1H), δ 3.33 (m, 1H), δ 3.64 (m, 1H), δ6.07 (s, 2H), δ 6.96-7.06 (m, 2H), δ 7.13 (d, 1H, J=1.3 Hz), δ 7.78 (d,1H, J=8.2 Hz), δ 7.88 (d, 2H, J=8.5 Hz), δ 7.94 (t, 1H, J=8.2 Hz), δ8.08 (d, 1H, J=8.2 Hz), δ 8.16 (d, 2H, J=8.5 Hz), δ 8.53 (s, 1H).

The compounds in the following table were synthesized as described aboveusing commercially available amines. Additional examples of theinvention were prepared following the above procedure withnon-substantial changes but using amines given in Table 5.

TABLE 5 Additional exemplary compounds of formula I. Compound No. Amine13 1-methylpiperazine 22 2,6-dimethylmorpholine 30piperidin-3-ylmethanol 34 2-(methylamino)ethanol 35(R)-pyrrolidin-2-ylmethanol 75 2-(pyrrolidin-1-yl)ethanamine 76pyrrolidine 90 piperidine 103 (tetrahydrofuran-2-yl)methanamine 109piperidin-4-ol 117 2-methylpropan-2-amine 118 cyclopentanamine 125(S)-2-(methoxymethyl)pyrrolidine 133 (R)-2-(methoxymethyl)pyrrolidine141 piperidin-4-ylmethanol 156 N-methylpropanamine 163 pyrrolidin-3-ol168 2-(2-aminoethoxy)ethanol 172 2-morpholinoethanamine 175furan-2-ylmethanamine 176 piperidin-3-ol 1782-(1-methylpyrrolidin-2-yl)ethanamine 180 3-methylpiperidine 182(S)-pyrrolidine-2-carboxamide 184 (R)-1-aminopropan-2-ol 1972-aminopropane-1,3-diol 199 2-amino-2-ethylpropane-1,3-diol 203N¹,N¹-dimethylethane-1,2-diamine 205 (R)-2-amino-3-methylbutan-1-ol 208cyclohexanamine 212 piperazin-2-one 232 2-aminoethanol 233piperidin-2-ylmethanol 234 2-(piperazin-1-yl)ethanol 244N-(cyclopropylmethyl)propan-1-amine 249 3-morpholinopropan-1-amine 2611-(piperazin-1-yl)ethanone 267 2-(1H-imidazol-4-yl)ethanamine 268(R)-2-aminopropan-1-ol 270 2-methylpiperidine 2732-(pyridin-2-yl)ethanamine 275 3,3-difluoropyrrolidine 2762-amino-2-methylpropan-1-ol 285 3-(1H-imidazol-1-yl)propan-1-amine 304piperidine-3-carboxamide 306 cyclobutanamine 307(S)-3-aminopropane-1,2-diol 311 N-methylcyclohexanamine 312N-methylprop-2-en-1-amine 316 2-amino-2-methylpropane-1,3-diol 325(5-methylfuran-2-yl)methanamine 330 3,3-dimethylbutan-1-amine 3322-methylpyrrolidine 335 2,5-dimethylpyrrolidine 336(R)-2-aminobutan-1-ol 338 propan-2-amine 339 N-methylbutan-1-amine 3424-amino-3-hydroxybutanoic acid 344 3-(methylamino)propane-1,2-diol 347N-(2-aminoethyl)acetamide 360 1-aminobutan-2-ol 364(S)-pyrrolidine-2-carboxylic acid 366 1-(2-methoxyethyl)piperazine 373(R)-2-aminopentan-1-ol

EE.1-Benzo[1,3]-dioxol-5-yl-N-[6-[4-[(methyl-methylsulfonyl-amino)methyl]phenyl]-2-pyridyl]-cyclopropane-1-carboxamide(Compound No. 292)

To the starting amine (brown semisolid, 0.100 g, ˜0.2 mmol, obtained bytreatment of the corresponding t-butyloxycarbonyl derivative bytreatment with 1N HCl in ether) was added dichloroethane (DCE) (1.5 mL),followed by the addition of pyridine (0.063 mL, 0.78 mmol) andmethansulfonyl chloride (0.03 mL, 0.4 mmol). The mixture was stirred at65° C. for 3 hours. After this time, LC/MS analysis showed ˜50%conversion to the desired product. Two additional equivalents ofpyridine and 1.5 equivalents of methansulfonyl chloride were added andthe reaction was stirred for 2 hours. The residue was concentrated andpurified by HPLC to yield1-benzo[1,3]dioxol-5-yl-N-[6-[4-[(methyl-methylsulfonyl-amino)methyl]phenyl]-2-pyridyl]-cyclopropane-1-carboxamide(0.020 g, 21% yield) as a white solid. ESI-MS m/z calc. 479.2. found480.1 (M+1)⁺.

FF.(R)-1-(3-hydroxy-4-methoxyphenyl)-N-(6-(4-(2-(hydroxymethyl)-pyrrolidin-1-ylsulfonyl)phenyl)pyridin-2-yl)cyclopropanecarboxamide

(R)-1-(3-(Benzyloxy)-4-methoxyphenyl)-N-(6-(4-(2-(hydroxymethyl)pyrrolidin-1-ylsulfonyl)phenyl)pyridin-2-yl)cyclopropanecarboxamide(28 mg, 0.046 mmol) was dissolved in ethanol (3 mL). Palladium oncharcoal (10%, 20 mg) was added and the reaction was stirred overnightunder 1 atm of hydrogen. The catalyst was filtered off and the productwas isolated by silica gel chromatography (50-80% EtOAc in hexane) toprovide(R)-1-(3-hydroxy-4-methoxyphenyl)-N-(6-(4-(2-(hydroxymethyl)pyrrolidin-1-ylsulfonyl)phenyl)pyridin-2-yl)cyclopropanecarboxamide(8 mg, 34%). ESI-MS m/z calc. 523.4. found 524.3 (M+1)⁺. Retention timeof 3.17 minutes.

2-Amino-5-phenylpyridine (CAS [33421-40-8]) is C-1.

GG.(R)-(1-(4-(6-Aminopyridin-2-yl)phenylsulfonyl)pyrrolidin-2-yl)methanolhydrochloride (C-2)

Step a: (R)-(1-(4-Bromophenylsulfonyl)pyrrolidin-2-yl)methanol

To a mixture of sat aq. NaHCO₃ (44 g, 0.53 mol), CH₂Cl₂ (400 mL) andpyrrolidin-2-yl-methanol (53 g, 0.53 mol) was added a solution of4-bromo-benzenesulfonyl chloride (127 g, 0.50 mol) in CH₂Cl₂ (100 mL).The reaction was stirred at 20° C. overnight. The organic phase wasseparated and dried over Na₂SO₄. Evaporation of the solvent underreduced pressure provided(R)-(1-(4-bromophenylsulfonyl)pyrrolidin-2-yl)methanol (145 g, crude),which was used in the next step without further purification. ¹H NMR(CDCl₃, 300 MHz) δ 7.66-7.73 (m, 4H), 3.59-3.71 (m, 3H), 3.43-3.51 (m,1H), 3.18-3.26 (m, 1H), 1.680-1.88 (m, 3H), 1.45-1.53 (m, 1H).

Step b:(R)-1-(4-Bromo-benzenesulfonyl)-2-(tert-butyl-dimethyl-silanyloxymethyl)pyrrolidine

To a solution of [1-(4-bromo-benzenesulfonyl)-pyrrolidin-2-yl]-methanol(50.0 g, 0.16 mol) and 1H-imidazole (21.3 g, 0.31 mol) in CH₂Cl₂ (500mL) was added tert-butylchlorodimethylsilane (35.5 g, 0.24 mol) inportions. After addition, the mixture was stirred for 1 hour at roomtemperature. The reaction was quenched with water (200 mL) and theseparated aqueous layer was extracted with CH₂Cl₂ (100 mL×3). Thecombined organic layers were washed with brine, dried over Na₂SO₄ andevaporated under vacuum to give1-(4-bromo-benzenesulfonyl)-2-(tert-butyldimethylsilanyloxymethyl)pyrrolidine(68.0 g, 99%). ¹H NMR (300 MHz, CDCl₃) δ 7.63-7.71 (m, 4H), 3.77-3.81(m, 1H), 3.51-3.63 (m, 2H), 3.37-3.43 (m, 1H), 3.02-3.07 (m, 1H),1.77-1.91 (m, 2H), 1.49-1.57 (m, 2H), 0.87 (s, 9H), 0.06 (d, J=1.8 Hz,6H).

Step c:(R)-4-(2-((tert-butyldimethylsilyloxy)methyl)pyrrolidin-1-ylsulfonyl)phenylboronicacid

To a solution of1-(4-bromo-benzenesulfonyl)-2-(tert-butyl-dimethyl-silanyloxymethyl)pyrrolidine(12.9 g, 29.7 mmol) and B(O^(i)Pr)₃ (8.4 g, 45 mmol) in dry THF (100 mL)was added dropwise n-BuLi (2.5 M in hexane, 29.7 mL) at −70° C. Afteraddition, the mixture was warmed slowly to −10° C. and treated with HCl(1M, 50 mL). The organic layer was separated and the aqueous layer wasextracted with ethyl acetate. The combined organic layers were driedover Na₂SO₄ and evaporated under vacuum. The organics were combined togive crude(R)-4-(2-((tert-butyldimethylsilyloxy)methyl)pyrrolidin-1-ylsulfonyl)phenylboronicacid (15.0 g), which was used directly in the next step.

Step d:(6-{4-[2-(tert-Butyl-dimethyl-silanyloxymethyl)-pyrrolidine-1-sulfonyl]phenyl}pyridin-2-yl)carbamicacid tert-butyl ester

To a solution of (6-bromo-pyridin-2-yl)carbamic acid tert-butyl ester(24.6 g, 90.0 mmol) in DMF (250 mL) were added(R)-4-(2-((tert-butyldimethylsilyloxy)-methyl)pyrrolidin-1-ylsulfonyl)phenylboronicacid (45.0 g), Pd(PPh₃)₄ (10.4 g, 9.0 mmol), potassium carbonate (18.6g, 135 mol) and water (200 mL). The resulting mixture was degassed bygently bubbling argon through the solution for 5 minutes at 20° C. Thereaction mixture was then heated at 80° C. overnight. DMF was removedunder vacuum. To the residue was added EtOAc (300 mL). The mixture wasfiltered through a pad of silica gel, which was washed with EtOAc (50mL×3). The combined organic extracts were evaporated under vacuum. Thecrude residue was purified by column (Petroleum Ether/EtOAc 20:1) togive(6-{4-[2-(tert-butyl-dimethyl-silanyloxymethyl)pyrrolidine-1-sulfonyl]phenyl}pyridin-2-yl)carbamicacid tert-butyl ester (22.2 g, 45% over 2-steps). ¹H NMR (300 MHz,CDCl₃) δ 8.09 (d, J=8.4 Hz, 2H), 7.88-7.96 (m, 3H), 8.09 (t, J=7.8 Hz,1H), 7.43-7.46 (m, 1H), 7.38 (s, 1H), 3.83-3.88 (m, 1H), 3.64-3.67 (m,1H), 3.53-3.59 (m, 1H), 3.41-3.47 (m, 1H), 3.08-3.16 (m, 1H), 1.82-1.91(m, 2H), 1.67-1.69 (m, 1H), 1.53-1.56 (m, 10H), 0.89 (s, 9H), 0.08 (d,J=2.4 Hz, 6H).

Step e:{6-[4-(2-Hydroxymethyl-pyrrolidine-1-sulfonyl)-phenyl]pyridin-2-ylcarbamicacid tert-butyl ester

A solution of crude(6-{4-[2-(tert-butyl-dimethyl-silanyloxymethyl)-pyrrolidine-1-sulfonyl]phenyl}-pyridin-2-yl)carbamicacid tert-butyl ester (22.2 g, 40.5 mmol) and TBAF (21.2 g, 81.0 mmol)in DCM (300 mL) was stirred at room temperature overnight. The mixturewas washed with brine (100 mL×3), dried over Na₂SO₄ and evaporated undervacuum to give{6-[4-(2-hydroxymethyl-pyrrolidine-1-sulfonyl)-phenyl]pyridin-2-yl}carbamicacid tert-butyl ester (15.0 g, 86%), which was used directly in the nextstep.

Step f:(R)-(1-(4-(6-Aminopyridin-2-yl)phenylsulfonyl)-pyrrolidin-2-yl)methanolhydrochloride (C-2)

A solution of{6-[4-(2-hydroxymethyl-pyrrolidine-1-sulfonyl)-phenyl]pyridin-2-yl}carbamicacid tert-butyl ester (15.0 g, 34.6 mmol) in HCl/MeOH (50 mL, 2M) washeated at reflux for 2 h. After cooling to room temperature, thereaction mixture was evaporated under vacuum and washed with EtOAc togive(R)-(1-(4-(6-aminopyridin-2-yl)phenylsulfonyl)pyrrolidin-2-yl)methanolhydrochloride (C-2; 11.0 g, 86%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.18 (d,J=8.7 Hz, 2H), 7.93-7.99 (m, 3H), 7.31 (d, J=7.2 Hz, 1H), 7.03 (d, J=8.7Hz, 1H), 3.53-3.57 (m, 2H), 3.29-35 (m, 2H), 3.05-3.13 (m, 1H),1.77-1.78 (m, 2H), 1.40-1.45 (m, 2H). MS (ESI) m/z (M+H)⁺334.2.

HH. N-(4-(6-Aminopyridin-2-yl)benzyl)methanesulfonamide (C-3)

Step a: [6-(4-Cyano-phenyl)-pyridin-2-yl]carbamic acid tert-butyl ester

A mixture of 4-cyanobenzeneboronic acid (7.35 g, 50 mmol),(6-bromo-pyridin-2-yl)carbamic acid tert-butyl ester (13.8 g, 50 mmol),Pd(Ph₃P)₄ (5.8 g, 0.15 mmol) and K₂CO₃ (10.4 g, 75 mmol) in DMF/H₂O(1:1, 250 mL) was stirred under argon at 80° C. overnight. DMF wasevaporated off under reduced pressure and the residue was dissolved inEtOAc (200 mL). The mixture was washed with water and brine, dried overNa₂SO₄, and concentrated to dryness. The residue was purified by column(Petroleum Ether/EtOAc 50:1) on silica gel to give[6-(4-cyano-phenyl)-pyridin-2-yl]carbamic acid tert-butyl ester (7.0 g,60%). ¹H NMR (300 MHz, CDCl₃) δ 8.02-8.07 (m, 2H), 7.95 (d, J=8.4 Hz,1H), 7.71-7.79 (m, 3H), 7.37-7.44 (m, 2H), 1.53 (s, 9H).

Step b: [6-(4-Aminomethyl-phenyl)-pyridin-2-yl]-carbamic acid ten-butylester

A suspension of [6-(4-cyano-phenyl)-pyridin-2-yl]carbamic acidtert-butyl ester (7.0 g, 24 mmol), Raney Ni (1.0 g) in EtOH (500 mL) andNH₃.H₂O (10 mL) was hydrogenated under H₂ (50 psi.) at 50° C. for 6 h.The catalyst was filtered off and the filtrate was concentrated todryness to give [6-(4-aminomethyl-phenyl)-pyridin-2-yl]-carbamic acidten-butyl ester, which was used directly in next step. ¹H NMR (300 MHz,CDCl₃) δ 7.83-7.92 (m, 3H), 7.70 (t, J=7.8 Hz, 1H), 7.33-7.40 (m, 4H),3.92 (brs, 2H), 1.53 (s, 9H).

Step c:{6-[4-(Methanesulfonylamino-methyl)-phenyl]-pyridin-2-yl}carbamic acidtert-butyl ester

To a solution of [6-(4-aminomethyl-phenyl)-pyridin-2-yl]-carbamic acidtert-butyl ester (5.7 g 19 mmol) and Et₃N (2.88 g, 29 mmol) indichloromethane (50 mL) was added dropwise MsCl (2.7 g, 19 mmol) at 0°C. The reaction mixture was stirred at this temperature for 30 min, andthen washed with water and brine, dried over Na₂SO₄ and concentrated todryness. The residue was recrystallized with DCM/Petroleum Ether (1:3)to give{6-[4-(methanesulfonylamino-methyl)-phenyl]-pyridin-2-yl}carbamic acidtert-butyl ester (4.0 g, 44% over two steps). ¹H NMR (300 MHz, CDCl₃) δ7.90-7.97 (m, 3H), 7.75 (t, J=8.4, 8.4 Hz, 1H), 7.54-7.59 (m, 1H),7.38-7.44 (m, 3H), 4.73 (br,1H), 4.37 (d, J=6.0 Hz, 2H), 2.90 (s, 3H),1.54 (s, 9H).

Step d: N-(4-(6-Aminopyridin-2-yl)benzyl)methane-sulfonamide (C-3)

A mixture of{6-[4-(methanesulfonylamino-methyl)-phenyl]-pyridin-2-yl}carbamic acidtert-butyl ester (11 g, 29 mmol) in HCl/MeOH (4M, 300 mL) was stirred atroom temperature overnight. The mixture was concentrated to dryness. Theresidue was filtered and washed with ether to giveN-(4-(6-aminopyridin-2-yl)benzyl)methane sulfonamide (C-3) (7.6 g, 80%)¹H NMR (300 MHz, DMSO-d₆) δ 14.05 (br s, 1H), 8.24 (br s, 2H), 7.91-7.98(m, 3H), 7.70 (t, J=6.0 Hz, 1H), 7.53 (d, J=8.1 Hz, 2H), 7.22 (d, J=6.9Hz, 1H), 6.96 (d, J=9 Hz, 1H), 4.23 (d, J=5.7 Hz, 2H), 2.89 (s, 3H). MS(ESI) m/z (M+H)⁺: 278.0,

II. 4-(6-Aminopyridin-2-yl)-N-methylbenzenesulfonamide hydrochloride(C-4)

Step a: 4-Bromo-N-methyl-benzenesulfonamide

To a mixture of sat aq. NaHCO₃ (42 g, 0.5 mol), CH₂Cl₂ (400 mL) andmethylamine (51.7 g, 0.5 mol, 30% in methanol) was added a solution of4-bromo-benzenesulfonyl chloride (127 g, 0.5 mol) in CH₂Cl₂ (100 mL).The reaction was stirred at 20° C. overnight. The organic phase wasseparated and dried over Na₂SO₄. Evaporation of the solvent underreduced pressure provided the 4-bromo-N-methyl-benzenesulfonamide (121g, crude), which was used in the next step without further purification.¹H NMR (CDCl₃, 300 MHz) δ 7.64-7.74 (m, 4H), 4.62-4.78 (m, 1H), 2.65 (d,J=5.4 Hz, 3H).

Step b: 4-(N-Methylsulfamoyl)phenylboronic acid

To a solution of 4-bromo-N-methyl-benzene sulfonamide (24.9 g, 0.1 mol)and B(O^(i)Pr)₃ (28.2 g, 0.15 mol) in THF (200 mL) was added n-BuLi (100mL, 0.25 mol) at −70° C. The mixture was slowly warmed to 0° C., then10% HCl solution was added until pH 3-4. The resulting mixture wasextracted with EtOAc. The organic layer was dried over Na₂SO₄, andevaporated under reduced pressure to give4-(N-methylsulfamoyl)phenylboronic acid (22.5 g, 96%), which was used inthe next step without further purification. ¹H NMR (DMSO-d₆, 300 MHz) δ8.29 (s, 2H), 7.92 (d, J=8.1 Hz, 2H), 7.69 (d, J=8.4 Hz, 2H), 2.36 (d,J=5.1 Hz, 3H).

Step c: tert-Butyl 6-(4-(N-methylsulfamoyl)phenyl)pyridin-2-ylcarbamate

To a solution of 4-(N-methylsulfamoyl)phenylboronic acid (17.2 g, 0.08mol) and (6-bromo-pyridin-2-yl)carbamic acid tert-butyl ester (21.9 g,0.08 mol) in DMF (125 mL) and H₂O (125 mL) were added Pd(PPh₃)₄ (9.2 g,0.008 mol) and K₂CO₃ (16.6 g, 0.12 mol). The resulting mixture wasdegassed by gently bubbling argon through the solution for 5 minutes at20° C. The reaction mixture was then heated at 80° C. for 16 h. Themixture was evaporated under reduced pressure, then poured into H₂O, andextracted with EtOAc. The organic phase was dried over Na₂SO₄, and wasevaporated under reduced pressure to give tert-butyl6-(4-(N-methylsulfamoyl)phenyl)pyridin-2-ylcarbamate (21 g, 58%), whichwas used in the next step without further purification.

Step d: 4-(6-Aminopyridin-2-yl)-N-methylbenzenesulfonamide hydrochloride

To a solution of tert-butyl6-(4-(N-methylsulfamoyl)phenyl)pyridin-2-ylcarbamate (8.5 g, 23.4 mmol)in MeOH (10 mL) was added HCl/MeOH (2M, 50 mL) at room temperature. Thesuspension was stirred at room temperature overnight. The solid productwas collected by filtration, washed with MeOH, and dried to give4-(6-aminopyridin-2-yl)-N-methylbenzenesulfonamide hydrochloride (5.0 g,71%). ¹H NMR (300 Hz, DMSO-d₆) δ 8.12 (d, J=8.4 Hz, 2H), 7.91-7.96 (m,3H), 7.58-7.66 (m, 1H), 7.31-7.53 (m, 1H), 7.27 (d, J=6.6, 1H), 6.97 (d,J=9.0, 1H), 2.43 (d, J=4.8 Hz, 3H). MS (ESI) m/z (M+H)⁺264.0.

The compounds in the following table were synthesized as described aboveusing commercially available or previously described carboxylic acidsand amines.

TABLE 6 Additional exemplary compounds of formula I. Compound No.Carboxylic acid Amine 164 A-9 C-1 165 A-3 C-2 169 A-17 C-3 170 A-3 C-4177 A-2 C-3 183 A-13 C-4 192 A-8 C-2 200 A-14 C-2 201 A-4 C-3 202 A-15C-2 211 A-15 C-3 214 A-6 C-2 218 A-2 C-4 220 A-4 C-2 221 A-10 C-2 223A-17 C-4 226 A-20 C-2 228 A-10 C-3 236 A-24 C-2 237 A-11 C-3 239 A-23C-2 240 A-11 C-4 242 A-13 C-2 245 A-15 C-4 246 A-8 C-3 248 A-13 C-3 250A-16 C-4 253 A-22 C-2 256 A-2 C-2 259 A-24 C-4 262 A-10 C-4 271 A-14 C-4279 A-19 C-2 281 A-16 C-2 282 A-8 C-4 284 A-17 C-2 302 A-5 C-2 317 A-10C-1 318 A-21 C-2 319 A-6 C-4 340 A-11 C-2 341 A-5 C-3 345 A-9 C-3 358A-18 C-2 362 A-16 C-3 363 A-5 C-4 369 A-9 C-4 372 A-9 C-2 376 A-35 C-2377 A-32 C-2 378 A-27 C-2 379 A-36 C-2 380 A-34 C-2 381 A-29 C-2 382A-28 C-2 383 A-25 C-2 384 A-30 C-2 385 A-33 C-2 386 A-31 C-2 387 A-37C-2 388 A-26 C-2 409 A-38 C-2 413 A-45 C-2

Physical data for examples of the invention are given in Table 7.

Additional exemplary compounds 164-388, as shown in Table 1, can also beprepared using appropriate starting materials and methods exemplifiedfor the previously described compounds.

TABLE 7 Physical data for exemplary compounds. Compound LCMS No. [M +H]⁺ LCMS RT NMR 1 416.3 2.39 2 442.5 2.7 3 427.1 4.1 4 508.3 3.43 5423.3 3.72 6 390.1 3.57 7 402.5 2.96 1H NMR (400 MHz, CD₃CN) δ 1.21-1.29(m, 2H), 1.62-1.68 (m, 2H), 3.05 (s, 6H), 6.06 (s, 2H), 6.86-6.97 (m,3H), 7.04-7.08 (m, 2H), 7.53-7.55 (m, 1H), 7.76-7.82 (m, 3H), 7.86 (t, J= 8.0 Hz, 1H), 8.34 (br s, 1H) 8 444.5 3.09 9 430.5 2.84 10 375.3 3.3911 403.5 2.83 12 390 3.14 14 520.2 1.38 15 387.3 3.71 16 389.3 2.9 17403.5 3.33 18 403.5 3.75 19 387.1 3.76 20 389 2.79 1H NMR (400 MHz,CD₃CN/ DMSO-d₆) δ 1.15-1.23 (m, 2H), 1.56-1.61 (m, 2H), 4.60 (s, 2H),6.05 (s, 2H), 6.94 (d, J = 8.3 Hz, 1H), 7.05-7.09 (m, 2H), 7.44 (d, J =8.2 Hz, 2H), 7.57-7.62 (m, 2H), 7.92 (s, 1H), 8.00 (dd, J = 2.5, 8.6 Hz,1H), 8.17 (d, J = 8.6 Hz, 1H), 8.48 (d, J = 1.8 Hz, 1H) 21 360 2.18 22387.3 3.77 23 535.2 2.81 24 464.1 2.35 1H-NMR (DMSO-d₆, 300 MHz) δ 8.40(s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.86 (m, 2H), 7.82 (m, 1H), 7.62 (d,J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 2.1 Hz, 1H),7.00 (m, 2H), 6.05 (s, 2H), 3.42 (m, 2H, overlap with water), 3.03 (m, J= 5.4 Hz, 2H), 2.98 (t, 1H), 1.49 (m, 2H), 1.14 (m, 2H). 25 403 3.29 1HNMR (400 MHz, CD₃CN/ DMSO-d₆) δ 1.14-1.17 (m, 2H), 1.52-1.55 (m, 2H),6.01 (s, 2H), 6.03 (s, 2H), 6.89-6.96 (m, 2H), 7.01-7.12 (m, 3H), 7.15(d, J = 1.8 Hz, 1H), 7.93 (dd, J = 8.7, 2.5 Hz, 1H), 8.05-8.11 (m, 2H),8.39-8.41 (m, 1H) 26 393 3.88 27 452.1 3.11 28 427.1 4.19 29 388.9 3.5830 375.3 2.95 31 535.2 2.42 32 359.1 3.48 33 394.9 3.77 34 360.3 2.96 35495.1 2.24 1H-NMR (300 MHz, CDCl₃) δ 8.22 (d, J = 8.7 Hz, 1H), 7.98 (m,3H), 7.80 (m, 3H), 7.45 (d, J = 7.5 Hz, 1H), 6.99 (dd, J = 8.1, 1.8 Hz,2H), 6.95 (d, J = 1.5 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.02 (s, 2H),3.77 (t, J = 5.1 Hz, 2H), 3.17 (m, J = 5.1 Hz, 2H), 2.85 (s, 3H), 1.70(q, J = 3.6 Hz, 2H), 1.19 (q, J = 3.6 Hz, 2H). 36 521.2 2.36 1H-NMR (300MHz, DMSO-d₆) δ 8.51 (s, 1H), 8.15 (d, J = 9.0 Hz, 2H), 8.06 (d, J = 8.4Hz, 1H), 7.92 (t, J = 7.8 Hz, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.76 (d, J= 7.5 Hz, 1H), 7.11 (d, J = 1.2 Hz, 1H), 7.03 (dd, J = 7.8, 1.8 Hz, 1H),6.97 (d, J = 7.8 Hz, 1H), 6.06 (s, 2H), 3.55 (m, 2H, overlap withwater), 3.15 (m, 2H), 3.07 (m, 1H), 1.77 (m, 2H), 1.50 (dd, J = 7.2, 4.5Hz, 2H), 1.43 (m, 2H), 1.15 (dd, J = 6.9, 3.9 Hz, 2H). 37 452.3 3.38 38398 3.02 39 483.1 2.58 1H-NMR (DMSO-d₆, 300 MHz) δ 10.01 (t, J = 6.0 Hz,1H), 8.39 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H),7.83 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 6.9 Hz, 1H), 7.33 (d, J = 8.4 Hz,2H), 7.11 (d, J = 2.1 Hz, 1H), 7.03 (d, J = 1.5 Hz, 1H), 6.99 (dd, 7.8Hz, 2H), 6.05 (s, 2H), 4.41 (d, J = 6 Hz, 2H), 1.48 (m, 2H), 1.14 (m,2H). 40 393.1 3.89 41 373.1 3.57 42 421.1 3.33 43 417.3 3.62 44 401.21.26 45 403.5 3.25 46 437.3 3.19 47 391.1 3.82 48 384.3 3.74 49 419.33.27 50 437 3.02 51 349 3.33 52 373.1 3.58 1H NMR (400 MHz, CD₃CN) δ1.17-1.20 (m, 2H), 1.58-1.61 (m, 2H), 2.24 (s, 3H), 6.01 (s, 2H), 6.90(d, J = 8.4 Hz, 1H), 7.04-7.06 (m, 2H), 7.16 (dd, J = 7.5, 0.8 Hz, 1H),7.23-7.33 (m, 4H), 7.79-7.89 (m, 2H), 8.10 (dd, J = 8.3, 0.8 Hz, 1H) 53387 3.62 54 394.1 3.06 55 419.3 2.92 56 407.5 3.55 57 388.9 2.91 58360.2 3.74 59 417.3 3.64 60 402.5 3.07 61 387.1 3.84 62 415.3 4.1 63 3843.35 64 360.3 3.58 65 465.1 2.47 1H-NMR (300 MHz, CDCl₃) δ 8.19 (d, J =8.1 Hz, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.92 (s, 1H), 7.89 (d, J = 8.4Hz, 2H), 7.76 (t, J = 7.5 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 6.99 (m,1H), 6.95 (br s, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.02 (s, 2H), 4.37 (t, J= 5.7 Hz, 1H), 3.02 (m, 2H), 1.70 (q, J = 3.9 Hz, 2H), 1.17 (q, J = 3.6Hz, 2H), 1.11 (t, J = 7.2 Hz, 3H). 66 401 3.24 67 393 3.88 68 407.5 4.0469 377.1 3.26 70 403.5 3.69 71 472.3 3.02 72 363 3.38 73 449.3 3.4 74416.3 2.43 75 373.1 3.69 76 534.2 1.36 77 491.2 2.7 78 384.3 3.72 79388.3 2.32 80 437.3 3.42 81 373 3.51 1H NMR (400 MHz, CD₃CN/ DMSO-d₆) δ1.07-1.27 (m, 2H), 1.50-1.67 (m, 2H), 2.36 (s, 3H), 6.10 (s, 2H), 6.92(d, J = 7.9 Hz, 1H), 7.01-7.09 (m, 2H), 7.28 (d, J = 7.9 Hz, 2H), 7.50(d, J = 8.2 Hz, 2H), 7.93-8.00 (m, 2H), 8.15 (d, J = 9.3 Hz, 1H), 8.44(d, J = 2.5 Hz, 1H) 82 419 2.71 1H NMR (400 MHz, CD₃CN) δ 1.29-1.32 (m,2H), 1.68-1.71 (m, 2H), 3.90 (s, 3H), 3.99 (s, 3H), 6.04 (s, 2H),6.70-6.72 (m, 2H), 6.93 (d, J = 8.4 Hz, 1H), 7.03-7.05 (m, 2H), 7.59 (d,J = 8.2 Hz, 1H), 7.73 (t, J = 7.6 Hz, 2H), 8.01 (t, J = 8.1 Hz, 1H),8.72 (br s, 1H) 83 417.3 3.41 84 394.9 3.74 85 401.3 3.97 86 473.5 2.6987 419.1 3.18 1H NMR (400 MHz, CD₃CN) δ 1.25-1.31 (m, 2H), 1.62-1.69 (m,2H), 3.84 (s, 3H), 3.86 (s, 3H), 6.04 (s, 2H), 6.62-6.70 (m, 2H), 6.92(d, J = 8.4 Hz, 1H), 7.00-7.08 (m, 2H), 7.30 (d, J = 8.3 Hz, 1H), 7.96(d, J = 8.9 Hz, 1H), 8.14 (dd, J = 8.9, 2.3 Hz, 1H), 8.38 (d, J = 2.2Hz, 1H), 8.65 (br s, 1H) 88 399 3.83 89 401.3 3.62 90 407.3 3.59 91505.2 2.88 92 384 3.36 1H NMR (400 MHz, CD₃CN) δ 1.27-1.30 (m, 2H),1.65-1.67 (m, 2H), 6.05 (s, 2H), 6.93 (d, J = 8.4 Hz, 1H), 7.04-7.09 (m,2H), 7.67 (t, J = 7.7 Hz, 1H), 7.79-7.81 (m, 1H), 7.91-7.94 (m, 1H),8.02-8.08 (m, 2H), 8.23 (dd, J = 8.9, 2.5 Hz, 1H), 8.50 (d, J = 1.9 Hz,1H), 8.58 (br s, 1H) 93 402 2.73 1H NMR (400 MHz, CD₃CN) δ 1.16-1.24 (m,2H), 1.57-1.62 (m, 2H), 6.05 (s, 2H), 6.95 (d, J = 7.6 Hz, 1H),7.05-7.09 (m, 2H), 7.71-7.75 (m, 2H), 7.95 (br s, 1H), 8.04-8.10 (m,3H), 8.22 (d, J = 8.7 Hz, 1H), 8.54 (d, J = 2.5 Hz, 1H) 94 419.3 2.8 95403.3 2.98 97 416.5 3.22 98 421 3 99 407.1 3.32 100 389 2.83 1H NMR (400MHz, CD₃CN) δ 1.21-1.26 (m, 2H), 1.60-1.65 (m, 2H), 4.65 (s, 2H), 6.03(s, 2H), 6.89-6.94 (m, 1H), 7.02-7.08 (m, 2H), 7.36-7.62 (m, 3H), 8.12(s, 2H), 8.36 (br s, 1H), 8.45-8.47 (m, 1H) 101 388.9 3.27 1H NMR (400MHz, CD₃CN) δ 1.22-1.24 (m, 2H), 1.61-1.63 (m, 2H), 3.82 (s, 3H), 6.04(s, 2H), 6.92 (d, J = 8.4 Hz, 1H), 7.04-7.12 (m, 4H), 7.34 (dd, J = 7.6,1.7 Hz, 1H), 7.38-7.43 (m, 1H), 8.03 (dd, J = 8.7, 2.3 Hz, 1H), 8.10(dd, J = 8.7, 0.7 Hz, 1H), 8.27 (br s, 1H), 8.37-8.39 (m, 1H) 102 401.33.77 103 430.5 3.04 104 388.3 2.32 105 521.2 2.46 106 393 3.63 107 4162.84 1H NMR (400 MHz, CD₃CN/ DMSO-d₆) δ 1.13-1.22 (m, 2H), 1.53-1.64 (m,2H), 2.07 (s, 3H), 6.08 (s, 2H), 6.90-6.95 (m, 1H), 7.01-7.09 (m, 2H),7.28 (d, J = 8.8 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.61 (d, J = 8.8 Hz,1H), 7.84 (d, J = 1.6 Hz, 1H), 7.95 (dd, J = 2.5, 8.7 Hz, 1H), 8.03 (brs, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.42 (d, J = 2.4 Hz, 1H), 9.64 (s, 1H)108 403.3 3.07 109 349.1 3.29 110 389.2 3.15 111 521.2 2.27 112 394 3.82113 407.5 3.3 114 417.1 3.17 115 398.1 3.22 116 394 3.1 1H NMR (400 MHz,CD₃CN) δ 1.18-1.26 (m, 2H), 1.59-1.64 (m, 2H), 6.05 (s, 2H), 6.95 (d, J= 8.4 Hz, 1H), 7.06-7.11 (m, 2H), 7.40 (d, J = 4.9 Hz, 1H), 7.92-7.96(m, 2H), 8.26 (d, J = 9.3 Hz, 1H), 8.36 (d, J = 1.7 Hz, 1H), 8.56 (d, J= 5.0 Hz, 1H), 8.70 (s, 1H) 117 363.3 3.48 118 374.3 3.54 119 494.3 3.59120 505.2 2.9 121 374.3 2.55 122 417.3 3.63 123 389.3 3.47 124 417.13.29 125 417.3 3.08 126 427.3 3.89 127 535.2 2.76 128 386.9 3.67 129377.1 3.67 130 389.1 3.4 1H NMR (400 MHz, CD₃CN) δ 1.22-1.24 (m, 2H),1.61-1.63 (m, 2H), 3.86 (s, 3H), 6.05 (s, 2H), 6.93 (d, J = 8.4 Hz, 1H),6.97-7.00 (m, 1H), 7.05-7.08 (m, 2H), 7.16-7.21 (m, 2H), 7.41 (t, J =8.0 Hz, 1H), 8.07-8.17 (m, 3H), 8.48-8.48 (m, 1H) 131 407.3 3.49 132 4193.09 1H NMR (400 MHz, CD₃CN) δ 1.17-1.25 (m, 2H), 1.57-1.64 (m, 2H),3.72 (s, 6H), 6.04 (s, 2H), 6.74 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4Hz, 1H), 7.05-7.08 (m, 2H), 7.35 (t, J = 8.4 Hz, 1H), 7.75 (d, J = 10.5Hz, 1H), 8.07-8.14 (m, 3H) 133 431.3 3.27 135 417.3 3.81 136 535.2 2.75137 403.5 3.35 138 432.5 2.76 H NMR (400 MHz, CD₃CN) δ 1.30-1.35 (m,2H), 1.69-1.74 (m, 2H), 3.09 (s, 6H), 4.05 (s, 3H), 6.04 (s, 2H), 6.38(d, J = 2.4 Hz, 1H), 6.50 (dd, J = 9.0, 2.4 Hz, 1H), 6.93 (d, J = 8.4Hz, 1H), 7.03-7.06 (m, 2H), 7.31 (d, J = 7.7 Hz, 1H), 7.71 (d, J = 8.8Hz, 2H), 7.97 (t, J = 8.3 Hz, 1H) 139 421.1 2.71 140 416.5 2.92 141 4102.83 1H NMR (400 MHz, CD₃CN) δ 1.28-1.37 (m, 2H), 1.66-1.73 (m, 2H),6.05 (s, 2H), 6.91-6.97 (m, 1H), 7.05-7.09 (m, 2H), 7.69-7.74 (m, 1H),7.82 (t, J = 7.7 Hz, 1H), 7.93 (d, J = 7.2 Hz, 1H), 8.04 (d, J = 8.8 Hz,1H), 8.15 (d, J = 8.2 Hz, 1H), 8.37 (d, J = 8.8 Hz, 1H), 8.58-8.65 (m,2H), 8.82 (br s, 1H), 8.94 (d, J = 6.2 Hz, 1H) 142 349.3 3.33 143 373.13.68 144 535.2 2.33 145 390.3 3.4 146 386.9 3.72 147 419.1 3.13 1H NMR(400 MHz, CD₃CN) δ 1.23-1.26 (m, 2H), 1.62-1.64 (m, 2H), 3.86 (s, 3H),3.89 (s, 3H), 6.04 (s, 2H), 6.93 (d, J = 8.4 Hz, 1H), 7.03-7.07 (m, 3H),7.17-7.19 (m, 2H), 8.06-8.15 (m, 2H), 8.38 (br s, 1H), 8.45-8.46 (m, 1H)148 393.1 3.72 1H NMR (400 MHz, CD₃CN) δ 1.20-1.27 (m, 2H), 1.58-1.67(m, 2H), 6.05 (s, 2H), 6.94 (d, J = 8.4 Hz, 1H), 7.05-7.09 (m, 2H),7.41-7.50 (m, 2H), 7.55-7.59 (m, 1H), 7.66-7.69 (m, 1H), 8.07 (d, J =11.2 Hz, 1H), 8.11 (br s, 1H), 8.16 (d, J = 8.8 Hz, 1H), 8.48 (d, J =1.9 Hz, 1H) 149 458.5 2.42 150 403.5 3.04 151 452.3 3.44 H NMR (400 MHz,MeOD) δ 1.30-1.36 (m, 2H), 1.71-1.77 (m, 2H), 2.58 (s, 3H), 6.04 (s,2H), 6.93 (dd, J = 0.8, 7.5 Hz, 1H), 7.04-7.08 (m, 2H), 7.86 (dd, J =0.8, 7.7 Hz, 1H), 8.00-8.02 (m, 2H), 8.08-8.12 (m, 3H), 8.19-8.23 (m,1H) 152 403 2.97 153 359.1 3.36 1H NMR (400 MHz, CD₃CN) δ 1.24-1.26 (m,2H), 1.62-1.65 (m, 2H), 6.05 (s, 2H), 6.93 (d, J = 8.4 Hz, 1H),7.05-7.08 (m, 2H), 7.42-7.46 (m, 1H), 7.49-7.53 (m, 2H), 7.63-7.66 (m,2H), 8.10-8.16 (m, 2H), 8.33 (br s, 1H), 8.48-8.48 (m, 1H) 154 395.13.34 155 393 3.7 156 390.2 3.7 157 403.5 3.33 158 390.2 3.58 159 493.22.85 160 411.3 3.94 161 419.1 3.2 162 488.1 3.62 163 438.1 3 164 314.13.38 165 538.5 3.28 166 466.1 2.9 167 429.3 2.95 168 526.3 3.189189 169498.3 3.7 170 468.3 3.27 171 444.5 2.24 172 551.1 2.849824 173 377 3.7174 493.9 2.69 175 517.9 3.423179 176 522.3 3.49262 177 502.1 3.43 178549.1 2.906129 179 480.1 2.51 180 520.3 4.295395 181 488.2 3.07 182535.1 3.267469 183 436.3 3.62 184 496.3 3.265482 185 403.5 2.88 186420.9 2.86 187 444.3 2.39 188 417.3 2.24 189 466.1 2.88 190 438.1 2.39191 401.1 3.44 192 552.3 3.18 193 452.3 2.55 194 415 4 195 479.1 1.08196 430.5 2.34 197 512.3 2.961206 198 444.5 2.75 H NMR (400 MHz,DMSO-d₆) δ 1.11-1.19 (m, 2H), 1.46-1.52 (m, 2H), 2.31 (s, 3H), 2.94 (s,3H), 2.99 (s, 3H), 6.08 (s, 2H), 6.97-7.05 (m, 2H), 7.13 (d, J = 1.6 Hz,1H), 7.35 (t, J = 1.5 Hz, 1H), 7.41 (t, J = 7.8 Hz, 2H), 7.51 (t, J =7.6 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 8.34(s, 1H) 199 540.3 3.18 200 520.3 3.79 201 452.3 3.22 202 536.5 3.63 203509.1 2.82 204 444.5 2.5 205 524.3 3.48 206 407.5 3.6 207 452.1 2.62 208520.3 4.06 209 416.1 2.3 210 452.3 2.8 H NMR (400 MHz, DMSO-d₆) δ1.11-1.19 (m, 2H), 1.47-1.52 (m, 2H), 2.31 (s, 6.08 (s, 2H), 6.96-7.07(m, 2H), 7.13 (d, J = 1.6 Hz, 1H), 7.43 (s, 1H), 7.57 (d, J = 8.1 Hz,2H), 7.69 (d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.2 Hz, 2H), 7.99 (d, J =8.4 Hz, 1H), 8.38 (s, 1H) 211 480.3 3.33 212 521.1 3.23 213 415.3 3.4214 562.3 3.71 215 403.3 2.67 216 421.1 2.91 217 387.1 2.89 218 488.33.73 219 403.7 2.43 220 508.5 3.46 221 508.3 3.46 222 401.1 2.76 223484.5 3.95 224 407.5 3.23 225 401.2 3.49 226 608.3 3.58 227 417.1 2.24228 452.3 3.21 229 407.1 3.08 230 401.3 2.68 231 389.1 2.36 232 481.93.155919 233 535.9 3.58 234 551.1 2.90 235 415.3 3.71 H NMR (400 MHz,DMSO-d₆) δ 1.12-1.17 (m, 2H), 1.23 (d, J = 6.9 Hz, 6H), 1.47-1.51 (m,2H), 2.30 (s, 3H), 2.92 (septet, J = 6.9 Hz, 1H), 6.08 (s, 2H),6.97-7.05 (m, 2H), 7.12-7.17 (m, 2H), 7.20-7.22 (m, 1H), 7.24-7.26 (m,1H), 7.36 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.95 (d, J =8.4 Hz, 1H), 8.32 (s, 1H) 236 540.3 3.85 237 456.5 3.35 238 416.5 2.35239 529.3 2.29 240 442.3 3.57 241 466.3 3.5 242 506.3 3.67 243 403.32.69 244 534.3 3.93 245 466.3 3.6 246 496.3 2.9 247 458.5 2.3 248 450.33.01 249 565.2 2.89 250 480.5 3.74 251 452.1 1.07 252 389.1 2.82 253530.3 2.8 254 466.1 1.06 255 488.2 3.05 256 558.3 3.46 257 407.5 3.27258 430.5 2.66 H NMR (400 MHz, DMSO-d₆) δ 1.12-1.18 (m, 2H), 1.47-1.54(m, 2H), 2.30 (s, 3H), 2.79 (d, J = 4.5 Hz, 3H), 6.08 (s, 2H), 6.96-7.07(m, 2H), 7.13 (d, J = 1.6 Hz, 1H), 7.48-7.57 (m, 2H), 7.70 (d, J = 8.4Hz, 1H), 7.78 (d, J = 1.5 Hz, 1H), 7.84 (dt, J = 7.3, 1.7 Hz, 1H), 7.98(d, J = 8.4 Hz, 1H), 8.36 (s, 1H), 8.50-8.51 (m, 1H) 259 470.3 3.82 260403.1 2.27 261 549.1 3.39 262 438.1 3.43 263 403.3 2.8 264 407.1 3.04265 430.5 2.18 266 403.3 2.96 267 531.9 2.81 268 496.3 3.24 269 373.52.76 270 520.3 4.21 271 450.3 3.77 272 403.2 1.09 273 543.1 2.89 274417.3 2.26 275 527.9 3.91 276 510.3 3.37 277 403.1 2.2 278 430.5 2.68 HNMR (400 MHz, DMSO-d₆) δ 1.12-1.19 (m, 2H), 1.47-1.51 (m, 2H), 2.31 (s,3H), 2.80 (d, J = 4.5 Hz, 3H), 6.08 (s, 2H), 6.97-7.05 (m, 2H), 7.13 (d,J = 1.6 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H),7.90 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 8.3 Hz, 1H), 8.35 (s, 1H), 8.50(q, J = 4.5 Hz, 1H) 279 536.5 3.19 280 480.3 3.25 281 550.5 3.78 282482.5 3.15 283 416.3 2.58 284 554.3 3.99 285 546.3 2.87 286 416.1 2.29287 443 4.02 288 466.3 2.76 289 373.1 2.84 290 429.3 3 291 403.1 2.24292 479.2 2.49 293 417.3 2.65 294 403.5 2.39 295 416.3 2.61 H NMR (400MHz, DMSO-d₆) δ 1.14-1.18 (m, 2H), 1.46-1.54 (m, 2H), 2.31 (s, 3H), 6.08(s, 2H), 6.97-7.05 (m, 2H), 7.13 (d, J = 1.6 Hz, 1H), 7.44 (s, 1H),7.49-7.56 (m, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.83-7.85 (m, 1H),7.87-7.91 (m, 1H), 7.99 (d, J = 8.4 Hz, 1H), 8.05 (s, 1H), 8.39 (s, 1H)296 387.1 3.09 297 430.2 2.38 298 403.2 2.72 299 387.3 2.86 300 387.33.03 301 403.5 2.44 302 508.3 3.45 303 417.3 2.58 304 549.1 3.35 305429.5 3.01 306 492.3 3.81 307 512.3 2.97 308 415.3 2.85 309 444.5 2.75310 430.5 2.41 311 534.3 3.92 312 492.3 3.99 313 387.3 2.84 314 430.52.37 315 387 1.12 316 526.3 3.08 317 344.2 3.35 318 536.5 3.17 319 492.33.69 320 430.2 2.38 321 452.3 2.55 322 387.1 2.6 323 387.1 3.01 324402.5 2.14 325 531.9 3.83 326 444.5 2.5 327 403.3 2.83 328 401.1 3.48329 415.3 3.36 330 522.3 4.14 331 387.1 3.01 332 505.9 4.06 333 417.12.58 334 403.5 2.92 335 520.3 4.22 336 510.3 3.36 337 401.1 2.73 338479.9 3.44 339 508.3 3.83 340 512.5 3.6 341 452.3 3.15 342 540.3 3.07343 480.3 3 344 526.3 3.15 345 422.1 3.21 346 415 4.05 347 523.1 3.10348 416.3 1.87 349 438.1 2.4 350 402.5 2.18 351 373.1 3.08 352 415.73.13 353 420.9 2.9 354 407.3 3.03 355 480.3 2.96 356 452.3 2.47 357466.3 2.63 358 536.5 3.26 359 402.1 2.2 360 510.3 3.42 361 407 3.11 362494.5 3.45 363 438.1 3.42 364 535.9 3.44 365 402.1 2.21 366 565.2 3.01367 403.5 2.36 368 444.5 2.97 369 408.5 3.43 370 403.3 2.45 371 430.52.43 372 478.3 3.47 373 524.3 3.50 374 466.3 2.35 375 416.5 2.36 376552.3 3.42 377 524.5 3.17 378 538.5 3.07 379 528.3 3.33 380 548.3 3.75381 526.3 3.46 382 520.5 3.48 383 518.1 3.55 384 542.3 3.59 385 550.53.69 386 524.3 3.15 387 522.5 3.78 388 542.2 3.6 389 467.3 1.93 390469.3 1.99 391 507.5 2.12 392 453.5 1.99 393 487.3 2.03 394 483.5 1.92395 441.3 4.33 396 453.3 1.93 397 439.5 1.94 398 471.3 2 399 537.5 2.1400 525.3 2.19 401 453.5 1.96 402 483.3 1.87 403 457.5 1.99 404 469.51.95 405 471.3 1.98 406 525.3 2.15 407 439.4 1.97 408 525.1 2.14 409618.7 3.99 410 374.5 2.46 411 507.5 2.14 412 390.1 3.09 413 552.3 4.04414 457.5 2.06 415 521.5 2.14 416 319 3.32 417 471.3 1.96 418 417.3 1.75419 473.3 2.04 420 389.3 2.94 421 457.5 1.99 422 467.3 1.96

Assays

Assays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompounds JJ. Membrane Potential Optical Methods for Assaying ΔF508-CFTRModulation Properties of Compounds

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

1. Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with F508-CFTR; a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hrs at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hrs at 27° C. to “temperature-correct” ΔF508-CFTR. The cells weresubsequently rinsed 3× with Krebs Ringers solution and loaded with thevoltage-sensitive dyes. To activate ΔF508-CFTR, 10 μM forskolin and theCFTR potentiator, genistein (20 μM), were added along with Cl⁻-freemedium to each well. The addition of Cl⁻-free medium promoted Cl⁻ effluxin response to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

2. Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. During the first addition, a Cl⁻-free medium withor without test compound was added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2-10 μM forskolin was added toactivate ΔF508-CFTR. The extracellular Cl⁻ concentration following bothadditions was 28 mM, which promoted Cl⁻ efflux in response to ΔF508-CFTRactivation and the resulting membrane depolarization was opticallymonitored using the FRET-based voltage-sensor dyes.

3. Solutions

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

Chloride-free bath solution: Chloride salts in Bath Solution #1 aresubstituted with gluconate salts. CC2-DMPE: Prepared as a 10 mM stocksolution in DMSO and stored at −20° C. DiSBAC₂(3): Prepared as a 10 mMstock in DMSO and stored at −20° C.

4. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1× NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hrs at 37° C. before culturing at 27° C. for24 hrs for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16-24hours

Electrophysiological Assays for Assaying ΔF508-CFTR ModulationProperties of Compounds 1. Using Chamber Assay

Using chamber experiments were performed on polarized epithelial cellsexpressing ΔF508-CFTR to further characterize the ΔF508-CFTR modulatorsidentified in the optical assays. FRT^(ΔF508-CFTR) epithelial cellsgrown on Costar Snapwell cell culture inserts were mounted in an Ussingchamber (Physiologic Instruments, Inc., San Diego, Calif.), and themonolayers were continuously short-circuited using a Voltage-clampSystem (Department of Bioengineering, University of Iowa, Iowa, and,Physiologic Instruments, Inc., San Diego, Calif.). Transepithelialresistance was measured by applying a 2-mV pulse. Under theseconditions, the FRT epithelia demonstrated resistances of 4 Ω/cm² ormore. The solutions were maintained at 27° C. and bubbled with air. Theelectrode offset potential and fluid resistance were corrected using acell-free insert. Under these conditions, the current reflects the flowof Cl⁻ through ΔF508-CFTR expressed in the apical membrane. The I_(SC)was digitally acquired using an MP100A-CE interface and AcqKnowledgesoftware (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

2. Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM) and the PDE inhibitor, IBMX (100 μM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 μM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing ΔF508-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 μM of the test compound for24 hours at 37° C. and were subsequently washed 3× prior to recording.The cAMP- and genistein-mediated I_(SC) in compound-treated cells wasnormalized to the 27° C. and 37° C. controls and expressed as percentageactivity. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(SC) comparedto the 37° C. controls.

3. Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane and was permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large Cl⁻ concentrationgradient across the epithelium. All experiments were performed 30 minafter nystatin permeabilization. Forskolin (10 μM) and all testcompounds were added to both sides of the cell culture inserts. Theefficacy of the putative ΔF508-CFTR potentiators was compared to that ofthe known potentiator, genistein.

4. Solutions

-   -   Basolateral solution (in mM): NaCl (135), CaCl₂ (1.2), MgCl₂        (1.2), K₂HPO₄ (2.4), KHP O₄ (0.6),        N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)        (10), and dextrose (10). The solution was titrated to pH 7.4        with NaOH.    -   Apical solution (in mM): Same as basolateral solution with NaCl        replaced with Na Gluconate (135).

5. Cell Culture

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR(FRT^(ΔF508-CFTR)) were used for Ussing chamber experiments for theputative ΔF508-CFTR modulators identified from our optical assays. Thecells were cultured on Costar Snapwell cell culture inserts and culturedfor five days at 37° C. and 5% CO₂ in Coon's modified Ham's F-12 mediumsupplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100μg/ml streptomycin. Prior to use for characterizing the potentiatoractivity of compounds, the cells were incubated at 27° C. for 16-48 hrsto correct for the ΔF508-CFTR. To determine the activity of correctionscompounds, the cells were incubated at 27° C. or 37° C. with and withoutthe compounds for 24 hours.

6. Whole-Cell Recordings

The macroscopic ΔF508-CFTR current (I_(ΔF508)) in temperature- and testcompound-corrected NIH3T3 cells stably expressing ΔF508-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5-6 MΩ when filled with the intracellular solution. Underthese recording conditions, the calculated reversal potential for Cl⁻(E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance >20 GΩ and a series resistance <15 MΩ. Pulse generation, dataacquisition, and analysis were performed using a PC equipped with aDigidata 1320 A/D interface in conjunction with Clampex 8 (AxonInstruments Inc.). The bath contained <250 μl of saline and wascontinuously perifused at a rate of 2 ml/min using a gravity-drivenperfusion system.

7. Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 μM forskolin and 20 μM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 μM of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 μM of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

8. Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in T_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated Ep_(Cl) (−28 mV).

9. Solutions

-   -   Intracellular solution (in mM): Cs-aspartate (90), CsCl (50),        MgCl₂ (1), HEPES (10), and 240 μg/ml amphotericin-B (pH adjusted        to 7.35 with CsOH).    -   Extracellular solution (in mM): N-methyl-D-glucamine (NMDG)-Cl        (150), MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35        with HCl).

10. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

11. Single-Channel Recordings

The single-channel activities of temperature-corrected ΔF508-CFTR stablyexpressed in NIH3T3 cells and activities of potentiator compounds wereobserved using excised inside-out membrane patch. Briefly, voltage-clamprecordings of single-channel activity were performed at room temperaturewith an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Allrecordings were acquired at a sampling frequency of 10 kHz and low-passfiltered at 400 Hz. Patch pipettes were fabricated from Corning KovarSealing #7052 glass (World Precision Instruments, Inc., Sarasota, Fla.)and had a resistance of 5-8 MΩ when filled with the extracellularsolution. The ΔF508-CFTR was activated after excision, by adding 1 mMMg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalyticsubunit (PKA; Promega Corp. Madison, Wis.). After channel activitystabilized, the patch was perifused using a gravity-drivenmicroperfusion system. The inflow was placed adjacent to the patch,resulting in complete solution exchange within 1-2 sec. To maintainΔF508-CFTR activity during the rapid perifusion, the nonspecificphosphatase inhibitor F⁻ (10 mM NaF) was added to the bath solution.Under these recording conditions, channel activity remained constantthroughout the duration of the patch recording (up to 60 min). Currentsproduced by positive charge moving from the intra- to extracellularsolutions (anions moving in the opposite direction) are shown aspositive currents. The pipette potential (V_(p)) was maintained at 80mV.

Channel activity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

12. Solutions

-   -   Extracellular solution (in mM): NMDG (150), aspartic acid (150),        CaCl₂ (5), MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with        Tris base).    -   Intracellular solution (in mM): NMDG-Cl (150), MgCl₂ (2), EGTA        (5), TES (10), and Tris base (14) (pH adjusted to 7.35 with        HCl).

13. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

The exemplified compounds of Table 1 have an activity with a range ofabout 100 nM and 20 μM as measured using the assays describedhereinabove. The exemplified compounds of Table 1 are found to besufficiently efficacious as measured using the assays describedhereinabove.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-65. (canceled)
 66. A method of treating or lessening the severity of cystic fibrosis in a patient, wherein the patient has a defective gene that causes a deletion of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator amino acid sequence, said method comprising the step of administering to the patient an effective amount of the compound

or a pharmaceutically acceptable salt thereof. 67-69. (canceled)
 70. The method of claim 66, wherein the patient has two copies of the defective gene.
 71. A method of treating or lessening the severity of cystic fibrosis in a patient, wherein the patient has a defective gene that causes a deletion of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator amino acid sequence, said method comprising the step of administering to said patient an effective amount of a composition comprising: i) the compound:

or a pharmaceutically acceptable salt thereof; and ii) a pharmaceutically acceptable carrier.
 72. The method of claim 71, wherein the patient has two copies of the defective gene.
 73. The method of claim 71 or 72, wherein the composition further comprises a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator, or a nutritional agent.
 74. The method of claim 71 or 72, wherein the composition further comprises a CFTR modulator.
 75. The method of claim 71 or 72 further comprising the additional step of administering to the patient a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator, or a nutritional agent.
 76. The method of claim 71 or 72, wherein the method comprises the additional step of administering to the patient a CFTR modulator.
 77. The method of claim 76, wherein the compound

is administered prior to, concurrently with, or subsequent to the CFTR modulator.
 78. The method of claim 77, wherein the compound is administered concurrently with the CFTR modulator.
 79. The method of claim 77, wherein the compound is administered prior to the CFTR modulator. 